Photochemical activation of surfaces for attaching biomaterial

ABSTRACT

A water-soluble photo-activatable polymer including: a photo-activatable group adapted to be activated by an irradiation source and to form a covalent bond between the water-soluble photo-activatable polymer and a matrix having at least one carbon; a reactive group adapted to covalently react with a biomaterial for subsequent delivery of the biomaterial to a cell; a hydrophilic group; and a polymer precursor. A composition including a monomolecular layer of the water-soluble photo-activatable polymer and a matrix having at least one carbon, wherein the monomolecular layer is covalently attached to the matrix by a covalent bond between the photo-activatable group and the at least one carbon. The composition further includes a biomaterial having a plurality of active groups, wherein the biomaterial is covalently attached to the monomolecular layer by covalent bonding between the active groups and reactive groups. Also provided is a method for delivery of a biomaterial to a cell.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Division of U.S. application Ser. No. 11/250,877,filed Oct. 14, 2005, which is a continuation in part of InternationalApplication No. PCT/US04/11861, filed Apr. 16, 2004, and claims prioritybenefit of U.S. Provisional Application No. 60/691,416, filed Jun. 17,2005, U.S. Provisional Application No. 60/545,127, filed Feb. 17, 2004,U.S. Provisional Application No. 60/546,233, filed Feb. 20, 2004, andU.S. Provisional Application No. 60/463,505, filed Apr. 16, 2003, theentire disclosures of all of which are incorporated herein by referencefor all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

Research leading to the disclosed invention was funded with funds fromthe National Heart Lung and Blood Institute under Contract No. HL59730.Accordingly, the United States government has certain rights in theinvention described herein.

BACKGROUND OF THE INVENTION

1. Field of Invention

This invention relates to surface modifications and more particularly toimmobilization of molecules to surfaces by photochemical coupling.

2. Description of Related Art

A common shortcoming of implantable medical devices or surfaces isrecognition of these devices by an organism as foreign objects followedby inflammation or even a rejection of such devices. Surfacemodification science concentrates on finding a better interface betweena living tissue and a solid matrix. It is known to use various coatingsto impart desirable properties to implantable surfaces. Such coatingsare based on polymers and may include biologically active materials.Challenges in preparing such coatings include attaching biologicallyactive materials to inert surfaces.

One of the techniques for derivatizing inert surfaces is photochemicalcoupling (see reference [1]). Photo-cross-linking chemistry based onorganic solvents is well established (see references [2-5]). It is notknown to use adsorption from aqueous solutions for application ofphoto-cross-linkers onto a polymer surface.

Aryl ketones (e.g., benzophenone or acetophenone derivatives) and arylazides are known as photo-activatable cross-linkers suitable forcovalent binding to virtually any type of polymer surface as describedby Amos et al. Upon irradiation with long-wave UV (at about 350 nm),benzophenone residues form energy-rich excited triplet species, whichare then inserted into carbon-hydrogen bonds (C—H bonds) of a polymericsurface by abstraction of hydrogen atoms from C—H bonds and form newcarbon-carbon bonds (C—C bonds), resulting in covalent binding ofbenzophenone residues onto the polymeric surface (see FIG. 1).

U.S. Pat. No. 5,071,909 to Pappin et al. discloses a method forimmobilizing proteins or peptides onto a membrane by formation of apolymeric network, which entraps the protein or peptide.

U.S. Pat. Nos. 3,959,078, 5,512,329 and 5,741,551 to Guire et al.disclose covalent bonding of polymeric molecules to surfaces throughexternal activation. This approach is used to bind fibronectin peptideto a polystyrene surface by a photo-reaction between the peptide and asurface having a photo-activatable group.

U.S. Pat. No. 5,637,460 to Swan et al. discloses attaching a targetmolecule (synthetic polymers, carbohydrates, proteins, lipids, nucleicacids, etc.) to a surface by using photo-activatable groups.

It is evident from the prior art discussed above that prior to thepresent invention, photo-cross-linkers were not used for the attachmentof functional reactive groups (other than the same photo-activatablegroups used in the initial step of photo-immobilization) to the surfacein order to activate it for further immobilization of biomolecules.

Replication defective adenoviruses (Ad or AdV) have been extensivelyused in both experimental and human gene therapy. Ad cell entry takesplace through receptor-mediated endocytosis via dualCoxsackie-Adenovirus Receptor (CAR)/αvβ3 integrins receptor system,although alternative pathways, such as fluid phase endocytosis might beoperative under certain conditions (Meier and Greber, 2003). Variouscell types differ widely in their level of CAR expression and this hasproved to be a limiting factor for the level of transgene expressionachievable with Ad. Furthermore, it is also clear that Ad cell entry andprocessing within cells via non-receptor endocytosis is a far lessefficient means for vector processing than CAR-mediated cell entry.Thus, transgene expression in cells with relatively low amounts of CARcan only be achieved with high doses of Ad (Baker, 2004; Xu et al.,2005), and this has proved to be a major concern in terms of safety andtoxicity (Yei et al., 1994; Thomas et al., 2001; Brunetti-Pierri et al.,2004).

Despite the foregoing developments, there is a need in the art foralternative means of attaching target molecules to surfaces.

All references cited herein are incorporated herein by reference intheir entireties.

BRIEF SUMMARY OF THE INVENTION

Accordingly, the invention provides a water-soluble photo-activatablepolymer comprising:

(a) a photo-activatable group, wherein the photo-activatable group isadapted to be activated by an irradiation source and to form a covalentbond between the water-soluble photo-activatable polymer and a matrixhaving at least one carbon;

(b) a reactive group, wherein the reactive group is adapted tocovalently react with a biomaterial;

(c) a hydrophilic group, wherein the hydrophilic group is present in anamount sufficient to make the water-soluble photo-activatable polymersoluble in water; and

(d) a polymer precursor.

In certain embodiments, the polymer precursor comprises at least onemonomer selected from the group consisting of allylamine, vinylamine,acrylic acid, carboxylic acid, alcohol, ethylene oxide, and acylhydrazine.

In certain embodiments, the reactive group is a member selected from thegroup consisting of an amino group, a thiol-reactive group, a carboxygroup, a thiol group, a protected thiol group, an acyl hydrazine group,an epoxy group, an aldehyde group, and a hydroxy group.

In certain embodiments, the thiol-reactive group is a member selectedfrom the group consisting of a 2-pyridyldithio group, a3-carboxy-4-nitrophenyldithio group, a maleimide group, an iodoacetamidegroup, and a vinylsulfonyl group.

In certain embodiments, the hydrophilic group is a member selected fromthe group consisting of an amino group and a carboxy group.

In certain embodiments, the photo-activatable group is a member selectedfrom the group consisting of an aryl ketone and an aryl azide. Incertain embodiments, the aryl ketone is a member selected from the groupconsisting of benzophenone and acetophenone.

In certain embodiments, the water-soluble polymer is represented by aformula:

wherein n is 50 to 2000 and k is 10 to 1000.

In certain embodiments, the water-soluble polymer is represented by aformula:

wherein n is 50 to 2000, k is 10 to 1000, and m is 10 to 1000.

Further provided is a process for producing the water-solublephoto-activatable polymer of the invention, the process comprising:

providing a polymer precursor comprising a plurality of reactive groupsand a plurality of hydrophilic groups;

providing a photo-activatable reagent; and

reacting the polymer with the photo-activatable reagent to obtain thewater-soluble photo-activatable polymer, wherein a first portion of thereactive groups and/or hydrophilic groups is modified with thephoto-activatable group.

In certain embodiments of the process, the first portion is from about1% to about 50% of the reactive groups and/or hydrophilic groups.

In certain embodiments, the first portion is about 20% of the reactivegroups and/or hydrophilic groups.

In certain embodiments, the process for producing the water-solublephoto-activatable polymer of the invention further comprises:

providing a reagent comprising a thiol-reactive group; and

reacting unmodified reactive groups and/or hydrophilic groups with thereagent to obtain the water-soluble polymer, wherein a second portion ofthe reactive groups and/or hydrophilic groups is modified with thethiol-reactive group.

In certain embodiments, a sum of the first portion and the secondportion is from about 60% to about 80%.

In certain embodiments, the thiol-reactive group is a member selectedfrom the group consisting of a 2-pyridyldithio group, a3-carboxy-4-nitrophenyldithio group, a maleimide group, an iodoacetamidegroup, and a vinylsulfonyl group.

Further provided is a composition of matter comprising a monomolecularlayer of the water-soluble photo-activatable polymer of the inventionand a matrix having at least one carbon, wherein the monomolecular layeris covalently attached to the matrix by a covalent bond between thephoto-activatable group and the at least one carbon.

In certain embodiments, the composition further comprises a biomaterialhaving a plurality of active groups, wherein the biomaterial iscovalently attached to the monomolecular layer by covalent bondingbetween the active groups and reactive groups.

In certain embodiments of the composition, at least one of the activegroups is a member selected from the group consisting of amine,carboxyl, hydroxyl, thiol, phenol, imidazole, and indole. Preferably,the at least one of the active groups comprise thiol.

In certain embodiments of the composition, the biomaterial is a memberselected from the group consisting of an antibody, a viral vector, agrowth factor, a bioactive polypeptide, a polynucleotide coding for thebio active polypeptide, a cell regulatory small molecule, a peptide, aprotein, an oligonucleotide, a gene therapy agent, a gene transfectionvector, a receptor, a cell, a drug, a drug delivering agent, nitricoxide, an antimicrobial agent, an antibiotic, an antimitotic, dimethylsulfoxide, an antisecretory agent, an anti-cancer chemotherapeuticagent, steroidal and non-steroidal anti-inflammatories, hormones, anextracellular matrix, a free radical scavenger, an iron chelator, anantioxidant, an imaging agent, and a radiotherapeutic agent. Preferably,the biomaterial is an anti-knob antibody, an adenovirus, a D1 domain ofthe Coxsackie-adenovirus receptor, insulin, an angiogenic peptide, anantiangiogenic peptide, avidin, biotin, IgG, protein A, transferrin, anda receptor for transferrin.

In certain embodiments of the composition, the matrix is a memberselected from a group consisting of polyurethane, polyester, polylacticacid, polyglycolic acid, poly(lactide-co-glycolide),poly(ε-caprolactone), polyethyleneimine, polystyrene, polyamide, rubber,silicone rubber, polyacrylonitrile, polyacrylate, and polymetacrylate,poly(alpha-hydroxy acid), poly(dioxanone), poly(orthoester),poly(ether-ester), poly(lactone), polytetrafluoroethylene, organosilane,mixtures thereof and copolymers thereof.

In certain embodiments of the composition, the matrix further comprisesa magnetic field-responsive agent. In certain embodiments, the magneticfield-responsive agent is a superparamagnetic agent. Preferably, thesuperparamagnetic agent is a member selected from the group consistingof magnetite and maghemite nanocrystals as such, as aggregates or asdispersion in polymer from the list above.

In certain embodiments of the composition, the matrix is an implantabledevice. Preferably, the implantable device comprises at least one memberselected from the group consisting of polyurethane, polyester,polylactic acid, poly(lactide-co-glycolide), poly(ε-caprolactone),polyethyleneimine, polystyrene, polyamide, rubber, silicone rubber,polyacrylonitrile, polyacrylate, polymetacrylate,polytetrafluoroethylene, organosilane, mixtures thereof and copolymersthereof.

In certain embodiments of the composition, the matrix is a particlehaving a diameter of about 5 nm to about 10 microns. Preferably, theparticle comprises at least one member selected from the groupconsisting of polylactic acid, poly(lactide-co-glycolide),poly(ε-caprolactone), polyethyleneimine, mixtures thereof and copolymersthereof.

Further provided is a method of making the composition of the invention,the method comprising:

providing the matrix having at least one carbon;

providing an aqueous solution of the water-soluble photo-activatablepolymer having the photo-activatable group and the reactive group;

contacting the matrix with the aqueous solution; and

photo-activating the photo-activatable group by irradiation tocovalently attach the water-soluble polymer via the photo-activatablegroup to the matrix and thereby forming the monomolecular layer of thecomposition.

In certain embodiments of the method of making the composition of theinvention, the irradiation is performed at a wavelength from about 190to about 900 nm. Preferably, the irradiation is performed at awavelength of 280 to 360 nm.

Additionally, certain embodiments of the method further compriseproviding a biomaterial having a plurality of active groups and reactingthe plurality of active groups with the water-soluble photo-activatablepolymer to covalently attach the biomaterial to the matrix.

Also provided is a process for delivery of a biomaterial, the processcomprising:

providing the composition of the invention as a monomolecular layer anda matrix having at least one carbon, wherein the monomolecular layer iscovalently attached to the matrix by a covalent bond between thephoto-activatable group and the at least one carbon;

providing a biomaterial having a plurality of active groups, wherein thebiomaterial is covalently attached to the monomolecular layer bycovalent bonding between the active groups and the reactive groups; and

administering the matrix to the cell and thereby delivering thebiomaterial.

Replication-defective adenoviral vectors have shown promise as a toolfor gene delivery-based therapeutic applications due to a number ofunique advantages, including an efficient mechanism of nuclear entry andability to transduce both quiescent and dividing cells in vivo withtransgenes of more than 30 kb without integration into the host cellgenome. Their use is however limited by their low efficacy in cellscharacterized by scarce expression of Coxsackie-adenovirus receptor(CAR), the primary receptor responsible for the cell entry of the virus,and by systemic adverse reactions resulting from the inability toeffectively localize and provide the sustained presence of the vector inthe target tissue, while minimizing its escape from the delivery site.To address these problems, inventors investigated the affinityimmobilization of adenovirus (AdV) on a biodegradable nanoparticle (NP)platform. Stable vector-specific association of AdV with nanoparticles(NP) leads to formation of composites that enter cells through aCAR-independent pathway allowing for use of substantially lower vectordoses for efficient transduction. Additionally the dissemination of thevector may be prevented by its association with 300-500 nm sized NPexhibiting lower rates of diffusion across solid tissues.

Biodegradable polyester-based nanoparticles possess highbiocompatibility, and can be prepared with strictly controlled size andnarrow size distribution, a prerequisite for safe parenteraladministration. Such particles have been extensively investigated asinjectable drug and gene carriers. The enhancement of the viral genedelivery by forming an affinity complex with biodegradable nanoparticlespresent a novel approach for improving and extending the applicabilityof the viral gene therapeutic strategies.

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

The invention will be described in conjunction with the followingdrawings in which like reference numerals designate like elements andwherein:

FIG. 1 is a reaction scheme illustrating binding of benzophenonecross-linkers to polymers having C—H bonds.

FIG. 2 is a reaction scheme demonstrating synthesis of polymericmulti-point benzophenone modifiers.

FIG. 3 is a reaction scheme depicting immobilization of thiol-containingproteins on a polymeric surface.

FIG. 4A demonstrates A10 cell growth inhibitory effect usingnanoparticles modified with D1 domain of CAR and adenoviral vectorencoding inducible nitric oxide synthase (iNOS-AdV).

FIG. 4B demonstrates A 10 cell growth inhibitory effect usingnanoparticles modified with IgG and iNOS-AdV.

FIG. 4C demonstrates A10 cell growth inhibitory effect usingnanoparticles modified with D1 and GFP-AdV.

FIG. 4D demonstrates A10 cell growth inhibitory effect usingnanoparticles modified with D1 and null-AdV. A null adenovirus is onethat contains no transgene.

FIG. 4E demonstrates GFP fluorescence (in relative units) ofnanoparticles modified with D1 and GFP-AdV.

FIG. 4F demonstrates GFP fluorescence of nanoparticles modified with IgGand GFP-AdV.

The A10 cell growth inhibitory effect of _(iNOS) Ad in the presence ofNP modified with D1 (_(D1)NP, FIG. 4A) and non-immune IgG (_(aIgG)NP,FIG. 4B) in comparison to the reporter gene expression mediated byrespective formulations prepared with _(GFP)Ad vector (FIGS. 4E, 4F).A10 growth inhibition was also studied for control formulationsincluding NP-Ad formulated with _(GFP)Ad (C) and _(NULL)Ad (FIG. 4D).The reporter gene expression and cell growth inhibition were assayed 48hr post treatment by direct cell fluorimetry (λ_(em)/λ_(ex) 485 nm/535nm) and using the Alamar Blue assay (λ_(em)/λ_(ex) 544 nm/580 nm),respectively.

FIG. 5 is a schematic representation of the method of preparing AdVimmobilized nanoparticles (AdV-NP).

FIGS. 6A-7C show the effect of knob fiber protein on the transductionand uptake of composite-forming nanoparticles (NP) measured as afunction of NP dose in smooth muscle cells (A10). The cells werepretreated with knob dissolved in the medium at 5 μg/ml for 1 hr priorto addition of Ad (2×10⁸/well) in the presence of _(D1)NP (0-4 μgPLA/well), or _(nIgG)NP used as a control. Knob-containing cell mediumwas aspirated, the cells were washed with PBS and incubated for 2 hrwith the formulations. Gene expression was assayed fluorimetrically(λ_(em) and λ_(ex) 485 nm and 535 nm, respectively) in live cells 2 dayspost treatment (FIG. 6A). The comparative effect of knob on the genetransfer in the presence of the complexes and the control nIgGNP,expressed as inhibition of expression, is presented in (FIG. 6B). Thecomparative effect of knob pretreatment on the intracellular level of NP(FIG. 6C) was measured using λ_(em)/λ_(ex) 544 nm/580 nm. Adimmobilization on NP resulted in a significantly greater expression(p<0.001, FIG. 6A). Error bars indicate standard deviation.

FIGS. 7A-7F are graphs demonstrating levels of GFP transgene expressionfollowing NP-Ad complex administration in cultures of rat aortic smoothmuscle cells (A10) (FIGS. 7A, 7D), sheep blood outgrowth endothelialcells (BOEC) (FIGS. 7B and 7E) and murine heart endothelioma cells (H₅V)(FIGS. 7C and 7F). Increasing amounts of NP were combined withGFP-encoding Ad at a dose of 1.3×10⁸ viral particles/well. FIGS. 7A-7Cshow GFP amount in cells 72 hr post treatment as a function of NP doseused to form the complexes with D1 tethering associated withsignificantly greater GFP expression for all NP doses (p<0.001). FIGS.7D-7F show GFP amount in cells treated with complexes at a NP dose of1.6 μg PLA/well as a function of time post treatment with D1 tetheringdemonstrating significantly greater expression at all time points(p<0.001). Error bars indicate standard deviation.

FIG. 8A is a transmission electron micrograph of a free NP and FIG. 8Bis a transmission electron micrograph of a NP-Ad complex formed viatethering Ad to the surface of PLA nanoparticles using D1-Ad affinitybinding. Electron microscopy (FEI Tecnai G2 Electron Microscope,Netherlands) was performed after negative staining with 2% (w/v) uranylacetate. The original magnification is ×50000.

FIGS. 9A and 9B are graphs demonstrating the uptake and retention ofcomplex-forming NPs and control NPs (_(D1)NP and _(nIgG)NP,respectively) by BOEC, A10 and H5V cells. The NPs were incubated withAdV at a dose 1.3×10⁸ viral particles/well and added to the cells for 2hr. The NP uptake (FIG. 9A) was determined 24 hr post treatment as afunction of the NP dose (0-3.2 μg PLA/well). The change in theintracellular levels with time (24-72 hr) was determined for NP appliedat a dose of 1.6 μg PLA/well PLA (FIG. 9B). The measurements wereperformed fluorimetrically in live cells using λ_(em)/λ_(ex) 544 nm/580nm Error bars indicate standard deviation.

FIG. 10A shows the effect of NP surface modification with D1 usingsurface activation withpoly(allylamine)-benzophenone-pyridyldithio-carboxylate (PBPC) andpoly(allylamine)-benzophenone-maleimido-carboxylate (PBMC) (see alsoFIG. 14). Thiolated biomolecule (e.g., HS-Protein) reacts with the NPsurface activated with PDT groups and MI groups to form a biodegradabledisulfide (S—S), or a non-biodegradable thioether (C—S) bond,respectively. The respective NP were incubated with Ad at a dose of1.3×10⁸ viral particles/well and added to the cells for 2 hr.

FIGS. 10B and 10C are graphs of NP uptake and GFP expression of thecomplexes respectively. The effect of the bond character on NP uptakeand GFP expression of the complexes was measured fluorimetrically usingλ_(em)/λ_(ex) 544 nm/580 nm, and 485 nm/535 nm, respectively, using_(nIgG)NP as a control.

FIG. 10D is a graph demonstrating a time course of the gene expressionmediated by the complexes formed at a NP dose of 1.6 μg PLA/well wasfollowed for 72 hr. Error bars indicate standard deviation.

FIGS. 11A and 11B are 3D graphs demonstrating GFP expression as afunction of NP and AdV dose with NP-Ad complexes employing anti-knob Ab(_(AK)NP-Ad) vs. D1 (_(D1)NP-Ad) as vector tethering agentsrespectively. The gene expression was assayed in A10 cells 48 hr posttreatment.

FIG. 12 shows micrographs demonstrating NP-Ad uptake and GFP expressionin A10 cells employing D1 (_(D1)NP-Ad, first column, A, D, and G) andanti-knob Ab (_(AK)NP-Ad, second column, B, E, and H) with free _(GFP)Ad(third column, C, F, and I) included as a control. The respective NP ata dose of 4.0 μg PLA/well were incubated with Ad at a dose 3.6×10⁸ viralparticles/well and added to the cells for 2 hr. Free Ad was included asa control (third column). The upper row shows the cell uptake of thecomplexes visualized using red fluorescent-labeled NP (A, B) and therespective GFP expression (D, E) observed 48 hr post treatment. Theperinuclear localization pattern and the substantially higher amount ofthe red fluorescence associated with _(D1)NP-Ad vs. _(AK)NP-Ad complexes(A vs. B) that corresponds to a qualitatively higher level of GFPexpression in _(D1) NP-Ad-treated cells (D vs. E) was observed. Theabsence of red fluorescence in the cells treated with free Ad (C) wasobserved. The A10 cells treated with NP-Ad and the free Ad exhibited nochange in their characteristic morphology (G-I) consistent with a lackof toxic effects. The original magnification was ×100.

FIG. 13 is a bar graph demonstrating luciferase (LUC) gene expression invivo in rat subcutaneous injection studies. _(D1)NP-Ad complexes wereformed by incubating _(LUC)Ad with _(D1)NP for 60 minutes followed byinjection in rats at a dose 8×10⁹ Ad and 150 μg PLA in 100 μl suspensionper animal (n=4). Five control animals received an identical dose offree _(LUC)Ad. Luciferase expression was measured in vivo assayingbioluminescence at 1 and 5 days post treatment (A). A significantlygreater LUC expression was observed with NP-_(LUC)Ad than _(LUC)Ad atone day (p=0.016), but not 5 days. Representative bioluminescence imagestaken at 1 day time point (not shown) demonstrate focal luciferase geneexpression with characteristic concentrical distribution around theinjection site with more intense LUC activity in the NP-_(LUC)Ad groupat 1 day.

FIG. 14 is a scheme demonstrating synthesis of photoreactive polymers:poly(allylamine)-benzophenone-pyridyldithio-carboxylate (PBPC) andpoly(allylamine)-benzophenone-maleimido-carboxylate (PBMC).

FIGS. 15 A-C are hysteresis curves for magnetic nanoparticles (FIG.15A), 3.5 mm long segment of Palmaz-Shatz stent wire (FIG. 15B), and 3.5mm long segment of Palmaz-Shatz stent wire coated with Nickel/Cobaltalloy (FIG. 15C). The linear region of the curve below 1000 Oerstads wasused to determine the susceptibility of the nanoparticles and thenormalized magnetic moment per unit length of the wires.

FIGS. 16A-C are graphs demonstrating simulated capture profiles ofmagnetic nanoparticles on implant materials as a function of time,applied field strength, and nanoparticle diameter. The capturepercentage of 380 nm diameter magnetic nanoparticles on unmodifiedPalmaz-Shatz 316L stent wire as a function of the applied field is shownin FIG. 16A, whereas the capture percentage on Nickel/Cobalt coatedPalmaz-Shatz 316L stent wire under similar conditions is shown in FIG.16B. The capture percentage on unmodified Palmaz-Shatz 316L stent wirein 1000 Oerstad fields as a function of nanoparticle diameter is shownin FIG. 16C. In each graph, the inlay shows the capture dynamics overthe first 100 milliseconds, indicating the potential for capturingparticles by intravenous injection in normal blood flow.

FIGS. 17A-17F are fluorescent images demonstrating the particle captureon 316L stent wires. FIGS. 17A-B show the nanoparticle accumulation onunmodified 316L stents without exposure to external field. FIGS. 17 C-Dshow the nanoparticle accumulation on unmodified 316L stents subjectedto a 1000 Oerstad external field. FIGS. 17 E-F show the nanoparticleaccumulation on Nickel/Cobalt coated 316L stents subjected to a 1000Oerstad external field. All scale bars were 100 mm.

FIGS. 18A-B are micrographic images of GFP-adenovirus studies withmagnetically responsive nanoparticle-adenoviral vector complexes,wherein FIG. 18A shows a closeup image of the transduction pattern in anA10 cell monolayer coated on a 316 Stainless Steel Electron Microscopygrid. The magnetic nanoparticles (red fluorescent) are co-localized inthe GFP positive cells that are positioned on the mesh wires. A largearea image of the transduction pattern in as shown in FIG. 18B confirmsthat the magnetic force direction is responsible for transfection ofcells only nearby the mesh. All scale bars are 100 μm. FIG. 18C is agraph demonstrating gene expression over the course of seven days. Allcontrols are negligible in comparison to the experimental group (blackline).

FIG. 19A is quantitative data documenting magnetically driven GFPexpression in cell culture via the use of magnetically responsivenanoparticles with tethered GFP-adenoviruses in the presence of a field(versus all of the relevant controls) using the techniques illustratedin FIGS. 18A-C.

FIG. 19B demonstrates minimal GFP expression in a rat carotid arterywith a steel coil deployed and injected with magnetically responsivenanoparticles with tethered GFP-adenoviruses, but without the use of amagnetic field, demonstrating little GFP expression.

FIG. 19C demonstrates minimal GFP expression in a rat carotid arterywith a steel coil deployed and injected with magnetically responsivenanoparticles with tethered GFP-adenoviruses, but with the use of amagnetic field demonstrating extensive GFP expression.

FIG. 20 is a schematic representation of the method of deliveringmagnetic nanoparticles carrying GFP encoding adenovirus to magneticfield gradients produced by 316L steel. The therapy (green glow) isdelivered only to the cells growing on top of the 316L stent wires, inorder to indicate the nature of local delivery method. The magneticfield across the mesh causes the development of high field gradientsbetween the mesh wires which attracts and binds magnetically responsivenanoparticles.

DETAILED DESCRIPTION OF THE INVENTION

The invention was driven by the desire to develop a composition and amethod for surface modification of inert surfaces useful forimplantation, which would permit attachment of molecular therapeuticssuch as proteins, genes, vectors, or cells and avoid using organicsolvents that can potentially damage both the surface and moleculartherapeutics. Moreover, utilizing therapeutic potential of site-specifictherapy (SST) with custom synthesized stent surfaces and heart valveleaflets, the present photochemical approach will permit surfaceactivation of abroad range of existing medical device configurations.

The inventors have discovered that biomaterial can be covalentlyattached to surfaces having at least one carbon by utilizing awater-soluble photo-activatable polymer of the invention which functionsas a multipoint polymeric cross-linker, wherein one function of thecross-linker is to photoimmobilize the water-soluble polymer to adesired surface and another function is to attach the desiredbiomaterial.

The water-soluble photo-activatable polymer of the invention comprises:

(a) a photo-activatable group, wherein the photo-activatable group isadapted to be activated by an irradiation source and to form a covalentbond between the water-soluble photo-activatable polymer and a matrixhaving at least one carbon;

(b) a reactive group, wherein the reactive group is adapted tocovalently react with a biomaterial;

(c) a hydrophilic group, wherein the hydrophilic group is present in anamount sufficient to make the water-soluble photo-activatable polymersoluble in water; and

(d) a polymer precursor.

The specific chemistry used herein has distinct advantages since itinvolves: 1) aqueous based exposures, thus removing any risk of damagingsurfaces that could be susceptible to organic solvent damage, and 2)addition of reactive groups, e.g., PDT groups, thus enabling sulfhydrylchemistry approaches for attaching linking proteins and peptides, suchas antibodies or receptor fragments.

A reaction between a thiol-reactive group (2-pyridyldithio, maleimide,etc.) attached to one protein molecule with a thiol group of anotherprotein molecule (or other biomolecule) is widely used for preparationof protein conjugates (See Greg T. Hermanson, Bioconjugate Techniques,Academic Press, San Diego 1996). Reaction of a thiol group with most ofthiol-reactive groups (particularly 2-pyridyldithio group) is veryselective and fast in aqueous media at mild conditions. Proteins can bethiolated using a partial reduction of disulfide bridges or viathiolation of lysine residues with a variety of reagents (see Hermanson,pp. 57-70). The reaction of thiolated proteins with polymeric surfacescontaining thiol-reactive groups would be ideal for the immobilizationof proteins on polymeric supports. At the same time, there is no methodfor providing polymeric surfaces with such thiol-reactive groups. Thepresent invention can efficiently solve this problem.

One of the reasons the surface-attachment of 2-pyridyldithio-groups(PDT-groups) to polymers via benzophenone photo-cross-linkers is notobvious is that PDT-groups are rather unstable and may not surviveconditions of photo-immobilization wherein active, energy-rich speciesappear.

The invention will now be described in more detail below.

The term “photo-activatable group” used herein denotes chemical groupscapable of generating active species such as free radicals, nitrenes,carbenes and excited states of ketons upon absorption of externalelectromagnetic or kinetic (thermal) energy. These groups may be chosento be responsive to various portions of the electromagnetic spectrum,i.e., the groups responsive to ultraviolet, visible and infraredportions of the spectrum. The preferred photo-activatable groups of theinvention are benzophenones, acetophenones and aryl azides. Uponexcitation, photo-activatable groups are capable of covalent attachmentto surfaces comprising at least one carbon such as polymers.

The water-soluble photo-activatable polymer of the invention may haveone or more photo-activatable groups. In certain embodiments, thewater-soluble photo-activatable polymers have at least onephoto-activatable group per molecule. Preferably, the water-solublephoto-activatable polymers have a plurality of photo-activatable groupsper molecule. More preferably, photo-activatable groups modify at least0.1% of monomeric units of a polymer precursor, even more preferably atleast 1%, and most preferably from about 20 to about 50%.

The irradiation source can be any source known in the art capable ofemitting the light having a wavelength absorbable by thephoto-activatable group of the invention. A UV-lamp is preferred whenthe benzophenone is used as the photo-activatable group.

The term “water-soluble polymer” as used in this disclosure means thatthe water-soluble photo-activatable polymer of the invention can bediluted with water to at least 1 wt % and preferably to at least 0.1 wt% to form a single phase at a temperature of 20° C., provided that wateris essentially free of an organic co-solvent.

The terms “surface” or “matrix” as used interchangeably herein mean asurface having at least one carbon. In a preferred embodiment of theinvention, the surface is a polymeric matrix. Other surfaces such asorganosylated materials (e.g., organosylated metals) can also be used inthe present invention.

The surface contemplated by the present invention can have any shape orform suitable for variety of purposes such as, for example, delivery ofa biomaterial to an organism. In that, the surface can be an existingmedical implant such as a stent or a cardiovascular valve, which can becovered with the composition of the invention. Also, the surface can befirst modified with either the water-soluble polymer of the invention orthe composition of the invention and then molded into the desired shape.Moreover, the surface can be in a form of polymeric particles.

Medical devices appropriate for the gene delivery system in the presentinvention include, but are not limited to, heart valves, wire sutures,temporary joint replacements and urinary dilators. Other suitablemedical devices for this invention include orthopedic implants such asjoint prostheses, screws, nails, nuts, bolts, plates, rods, pins, wires,inserters, osteoports, halo systems and other orthopedic devices usedfor stabilization or fixation of spinal and long bone fractures ordisarticulations. Other devices may include non-orthopedic devices,temporary placements and permanent implants, such as tracheostomydevices, jejunostomy and gastrostomy tubes, intraurethral and othergenitourinary implants, stylets, dilators, stents, vascular clips andfilters, pacemakers, wire guides and access ports of subcutaneouslyimplanted vascular catheters.

The polymeric matrix of the invention can be biodegradable andnon-biodegradable. Non-limiting examples of the polymeric matrix used inthe invention are poly(urethane), poly(ester), poly(lactic acid),poly(glycolic acid), poly(lactide-co-glycolide), poly(ε-caprolactone),poly(ethyleneimine), poly(styrene), poly(amide), rubber, siliconerubber, poly(acrylonitrile), poly(acrylate), poly(metacrylate),poly(alpha-hydroxy acid), poly(dioxanone), poly(orthoester),poly(ether-ester), poly(lactone), mixtures thereof and copolymers ofcorresponding monomers.

In certain embodiments of the composition, the matrix further comprisesa magnetic field-responsive agent. In certain embodiments, the magneticfield-responsive agent is a superparamagnetic agent. Preferably, thesuperparamagnetic agent is a member selected from the group consistingof magnetite and maghemite nanocrystals.

The water-soluble photo-activatable polymer of the invention comprises apolymer precursor and the following groups covalently attached to thepolymeric precursor: the photo-activatable group as described above, areactive group, and a hydrophilic group.

The polymeric precursor of the water-soluble photo-activatable polymerof the invention can be prepared using methods known in the art from apolymer (biodegradable or a non-biodegradable) comprising reactivegroups and hydrophilic groups, which is then modified to containphoto-activatable groups (e.g., see Example 1). Non-limiting examples ofsuch precursors are polymers containing monomers such as allylamine,vinylamine, acrylic acid, carboxylic acid, alcohol, ethylene oxide, andacyl hydrazine. Preferably, the polymer precursor is poly(allylamine) orpoly(acrylic acid). In certain embodiments of the invention, thepoly(allylamine) has a molecular weight of about 5 KDa to about 200 KDa.In the preferred embodiment, the molecular weight is from 15 KDa to 70KDa.

Also, it can be prepared by polymerization of monomeric blockscontaining the above groups. Such methods are also known in the art. Incertain embodiments of the invention, the polymeric precursor comprisesat least one monomer selected from the group consisting of allylamine,vinylamine, acrylic acid, carboxylic acid, alcohol, ethylene oxide, andacyl hydrazine.

The reactive group of the water-soluble photo-activatable polymer of theinvention is a chemical group which is selected for its ability tocovalently react with a biomaterial. A person skilled in the art wouldunderstand that if a thiol reactive group is selected as reactive groupof the water-soluble photo-activatable polymer, a corresponding thiolgroup should be selected for a reactive group in the biomaterial.Non-limiting examples of the reactive group are an amino group (primaryor secondary), a thiol-reactive group, a carboxy group, a thiol group, aprotected thiol group, an acyl hydrazine group, an epoxy group, analdehyde group, and a hydroxy group. Preferably, the thiol-reactivegroup is selected from the group consisting of a 2-pyridyldithio group,a 3-carboxy-4-nitrophenyldithio group, a maleimide group, aniodoacetamide group, and a vinylsulfonyl group.

The hydrophilic group of the water-soluble photo-activatable polymer ofthe invention is present in an amount sufficient to make thewater-soluble photo-activatable polymer soluble in water. In certainembodiments of the invention, the hydrophilic group is an amino group ora carboxy group.

The reactive group and the hydrophilic group of the water-solublephoto-activatable polymer of the invention can be identical ordifferent. In one embodiment of the invention, both the reactive groupand the hydrophilic group are amino groups. In another embodiment of theinvention, the reactive group is the 2-pyridyldithio group, and thehydrophilic group is the carboxy group.

In certain embodiments of the invention, the photo-activatable group isat least one of an aryl ketone and an aryl azide. Preferably, the arylketone is benzophenone or acetophenone.

In one embodiment of the invention, the water-soluble polymer ispoly(allylamine) based benzophenone (PAA-BzPh) and is represented by aformula:

wherein n is 50 to 2000 and k is 10 to 1000.

In another embodiment of the invention, the water-soluble polymer ispoly(allylamine) based benzophenone further modified to contain2-pyridyldithio groups (PDT-BzPh) and is represented by a formula:

wherein n is 50 to 2000, k is 10 to 1000, and m is 10 to 1000.

In yet another embodiment, the water-soluble photo-activatable polymerof the invention ispoly(allylamine)-benzophenone-pyridyldithio-carboxylate (PBPC) orpoly(allylamine)-benzophenone-maleimido-carboxylate (PBMC) (see FIG. 14and Example 9).

Methods of preparation of the water-soluble photo-activatable polymer ofthe invention based on poly(allylamine) and poly(acrylic acid) aredescribed in Examples 1 and 2.

The water-soluble photo-activatable polymer of the invention can beprepared by radical polymerization of a mixture of three types ofmonomers (e.g., acrylamide-based monomers), each containing only one ofthe groups, as shown below:

In this invention, identity of spacers is not crucial. X, Y and Z can beresidues of aliphatic, cyclo-aliphatic or aromatic hydrocarbons, whichmay include heteroatoms (O, N, S, etc.) and contain functional groupsnot interfering with both the polymerization and the further performanceof the resulting multifunctional polymeric cross-linker (like OH, amide,etc.).

The ratio between these groups in the final product can be controlled bychanging the ratio of monomers. Conditions for polymerization are knownto persons skilled in the art. Additionally, each monomer can bear two,or all the three types of the groups. Other types of polymerization orpolycondensation known in the art can also be used.

Upon excitation of photo-activatable groups, the water-solublephoto-activatable polymer of the invention is covalently bonded tocarbon atoms of the surface and forms a monomolecular layer on thesurface (see FIG. 1).

The term “layer” used herein means a contiguous and non-contiguousdeposit formed by a covalent bonding of water-soluble photo-activatablepolymers of the invention to the surface. Preferably, the layer ishighly homogeneous and consists essentially of the water-solublephoto-activatable polymer of the invention.

Further provided is a composition of matter comprising a monomolecularlayer of the water-soluble photo-activatable polymer of the inventionand a matrix having at least one carbon, wherein the monomolecular layeris covalently attached to the matrix by a covalent bond formed betweenthe photo-activatable group and the at least one carbon of the matrix.

In certain embodiments, the composition further comprises a biomaterialhaving a plurality of active groups, wherein the biomaterial iscovalently attached to the monomolecular layer by covalent bondingbetween active groups and reactive groups (see FIGS. 3, 5, and 10A).

In certain embodiments of the composition, at least one of the activegroups is a member selected from the group consisting of amine,carboxyl, hydroxyl, thiol, phenol, imidazole, and indole. Preferably, atleast one of the active groups is a thiol group.

Biomaterial

The biomaterial of the present invention can be any molecule ormacromolecule to which a suitable reactive group, such as a carboxy(—COOH), amino (—NH₂) or thiol group (—SH) is attached. For example,proteins or peptides that have been modified to comprise a thiol groupor comprise an amino group can be used. The biomaterial also has atherapeutic utility.

Suitable biomaterial include pharmaceuticals, nucleic acids, such astransposons, signaling proteins that facilitate wound healing, such asTGF-β, FGF, PDGF, IGF and GH proteins that regulate cell survival andapoptosis, such as Bcl-1 family members and caspases; tumor suppressorproteins, such as the retinoblastoma, p53, PAC, DCC. NF1, NF2, RET, VHLand WT-1 gene products; extracellular matrix proteins, such as laminins,fibronectins and integrins; cell adhesion molecules such as cadherins,N-CAMs, selectins and immunoglobulins; anti-inflammatory proteins suchas Thymosin beta-4, IL-10 and IL-12.

In certain embodiments, the biomaterial includes at least one ofheparin, covalent heparin, or another thrombin inhibitor, hirudin,hirulog, argatroban, D-phenylalanyl-L-poly-L-arginyl chloromethylketone, or another antithrombogenic agent, or mixtures thereof;urokinase, streptokinase, a tissue plasminogen activator, or anotherthrombolytic agent, or mixtures thereof, a fibrinolytic agent; avasospasm inhibitor; a calcium channel blocker, a nitrate, nitric oxide,a nitric oxide promoter or another vasodilator; an antimicrobial agentor antibiotic; aspirin, ticlopidine, a glycoprotein IIb/IIIa inhibitoror another inhibitor of surface glycoprotein receptors, or anotherantiplatelet agent; colchicine or another antimitotic, or anothermicrotubule inhibitor, dimethyl sulfoxide (DMSO), a retinoid or anotherantisecretory agent; cytochalasin or another actin inhibitor; aremodeling inhibitor; deoxyribonucleic acid, an antisense nucleotide oranother agent for molecular genetic intervention; methotrexate oranother antimetabolite or antiproliferative agent; tamoxifen citrate,Taxol or derivatives thereof, or other anti-cancer chemotherapeuticagents; dexamethasone, dexamethasone sodium phosphate, dexamethasoneacetate or another dexamethasone derivative, or anotheranti-inflammatory steroid or non-steroidal anti-inflammatory agent;cyclosporin or another immunosuppressive agent; trapidal (a PDGFantagonist), angiogenin, angiopeptin (a growth hormone antagonist), agrowth factor or an anti-growth factor antibody, or another growthfactor antagonist; dopamine, bromocriptine mesylate, pergolide mesylateor another dopamine agonist; radiotherapeutic agent; iodine-containingcompounds, barium-containing compounds, gold, tantalum, platinum,tungsten or another heavy metal functioning as a radiopaque agent; apeptide, a protein, an enzyme, an extracellular matrix component, acellular component or another biologic agent; captopril, enalapril oranother angiotensin converting enzyme (ACE) inhibitor; ascorbic acid,alpha tocopherol, superoxide dismutase, deferoxamine, a 21-amino steroid(lasaroid) or another free radical scavenger, iron chelator orantioxidant; a ¹⁴C—, ³H—, ³²P— or ³⁶S-radiolabelled form or otherradiolabelled form of any of the foregoing; a hormone; estrogen oranother sex hormone; AZT or other antipolymerases; acyclovir,famciclovir, rimantadine hydrochloride, ganciclovir sodium or otherantiviral agents; 5-aminolevulinic acid, meta-tetrahydroxyphenylchlorin,hexadecafluoro zinc phthalocyanine, tetramethyl hematoporphyrin,rhodamine 123 or other photodynamic therapy agents; an IgG2 Kappaantibody against Pseudomonas aeruginosa exotoxin A and reactive withA431 epidermoid carcinoma cells, monoclonal antibody against thenoradrenergic enzyme dopamine beta-hydroxylase conjugated to saporin orother antibody targeted therapy agents; gene therapy agents; andenalapril and other prodrugs, or a mixture of any of these.

Additionally, the biomaterial can be either component of anyaffinity-ligand pair. Examples of such affinity ligand pairs includeavidin-biotin and IgG-protein A. Furthermore, the biomaterial can beeither component of any receptor-ligand pair. One example is transferrinand its receptor. Other affinity ligand pairs include powerful hydrogenbonding or ionic bonding entities such as chemical complexes. Examplesof the latter include metallo-amine complexes. Other such attractivecomplexes include nucleic acid base pairs, via immobilizingoligonucleotides of a specific sequence, especially antisense. Nucleicacid decoys or synthetic analogues can also be used as pairing agents tobind a designed gene vector with attractive sites. Furthermore, DNAbinding proteins can also be considered as specific affinity agents;these include such entities as histones, transcription factors, andreceptors such as the gluco-corticoid receptor.

In one preferred embodiment, the biomaterial is an anti-nucleic acidantibody. The antibody can therefore specifically bind a nucleic acid,which encodes a product (or the precursor of a product) that decreasescell proliferation or induces cell death, thereby mitigating the problemof restenosis in arteries and other vessels. The nucleic acid that istethered to a matrix via the antibody can efficientlytransfect/transducer cells. In general terms, the field of “genetherapy” involves delivering into target cells some polynucleotide, suchas an antisense DNA or RNA, a ribozyme, a viral fragment, or afunctionally active gene, that has a therapeutic or prophylactic effecton the cell or the organism containing it (see Culver, 1994, GENETHERAPY: A HANDBOOK FOR PHYSICIANS (Mary Ann Liebert, Inc., New York,N.Y.)). The antibody of the composition can be a full-length (i.e.,naturally occurring or formed by normal immuno-globulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody, or IgM or any antibody subtype) or an immunologically active(i.e., specifically binding) portion of an immunoglobulin molecule. Theantibody comprises one or more sites which specifically bind with anucleic acid (i.e., which does not substantially bind other types ofmolecules). The binding site can be one which binds specifically with anucleic acid of a desired type without regard to the nucleotide sequenceof the nucleic acid. The binding site can, alternatively, be one whichbinds specifically only with a nucleic acid comprising a desirednucleotide sequence. Preferably, the antibody is a thiol modifiedantibody.

The complex formed between a polynucleotide and a cognate antibody canbe immobilized on a variety of surfaces such that, when the surface isexposed to a physiological environment in situ, the attachedpolynucleotide is released, over time, in a manner that enhancesdelivery of the polynucleotide to cells in the proximity. Surprisingly,DNA transfer by way of immunospecific tethering maintains the nucleicacid in regions that are subject to gene therapy.

Examples of suitable antibodies include Fv, F(ab), and F(ab′)₂fragments, which can be generated is conventional fashion, as bytreating an antibody with pepsin or another proteolytic enzyme. Thenucleic acid-binding antibody used in a composition of the presentinvention can be polyclonal antibody or a monoclonal antibody. A“monoclonal” antibody comprises only one type of antigen binding sitethat specifically binds with the nucleic acid. A “polyclonal” antibodycan comprise multiple antigen binding sites that specifically bind thenucleic acid. An antibody employed in this invention preferably is afull-length antibody or a fragment of an antibody, such as F(ab)₂, thatpossesses the desired binding properties.

A nucleic acid for use in the present invention can be anypolynucleotide that one desires to transport to the interior of a cell.In this context, a “therapeutic polynucleotide” is a polymer ofnucleotides that, when provided to or expressed in a cell, alleviates,inhibits, or prevents a disease or adverse condition, such asinflammation and/or promotes tissue healing and repair (e.g., woundhealing). The nucleic acid can be composed of deoxyribonucleosides orribonucleosides, and can have phosphodiester linkages or modifiedlinkages, such as those described below. The phrase “nucleic acid” alsoencompasses polynucleotides composed of bases other than the five thatare typical of biological systems: adenine, guanine, thymine, cytosineand uracil.

A suitable nucleic acid can be DNA or RNA, linear or circular and can besingle-or-double-stranded. The “DNA” category in this regard includescDNA; genomic DNA; triple helical, supercoiled, Z-DNA and other unusualforms of DNA; polynucleotide analogs; an expression construct thatcomprises a DNA segment coding for a protein, including a therapeuticprotein; so-called “antisense” constructs that, upon transcription,yield a ribozyme or an antisense RNA; viral genome fragments, such asviral DNA; plasmids and cosmids; and a gene or gene fragment.

The nucleic acid also can be RNA, for example, antisense RNA, catalyticRNA, catalytic RNA/protein complex (i.e., a “ribozyme”), and expressionconstruct comprised of RNA that can be translated directly, generating aprotein, or that can be reverse transcribed and either transcribed ortranscribed and then translated, generating an RNA or protein product,respectively; transcribable constructs comprising RNA that embodies thepromoter/regulatory sequence(s) necessary for the generation of DNA byreverse transcription; viral RNA; and RNA that codes for a therapeuticprotein, inter alia. A suitable nucleic acid can be selected on thebasis of a known, anticipated, or expected biological activity that thenucleic acid will exhibit upon delivery to the interior of a target cellor its nucleus.

The length of the nucleic acid is not critical to the invention. Anynumber of base pairs up to the full-length gene may be transfected. Forexample, the nucleic acid can be linear or circular double-stranded DNAmolecule having a length from about 100 to 10,000 base pairs in length,although both longer and shorter nucleic acids can be used.

The nucleic acid can be a therapeutic agent, such as an antisense DNAmolecule that inhibits mRNA translation. Alternatively, the nucleic acidcan encode a therapeutic agent, such as a transcription or translationproduct which, when expressed by a target cell to which the nucleicacid-containing composition is delivered, has a therapeutic effect onthe cell or on a host organism that includes the cell. Examples oftherapeutic transcription products include proteins (e.g., antibodies,enzymes, receptors-binding ligands, wound-healing proteins,anti-restenotic proteins, anti-oncogenic proteins, and transcriptionalor translational regulatory proteins), antisense RNA molecules,ribozymes, viral genome fragments, and the like. The nucleic acidlikewise can encode a product that functions as a marker for cells thathave been transformed, using the composition. Illustrative markersinclude proteins that have identifiable spectroscopic properties, suchas green fluorescent protein (GFP) and proteins that are expressed oncell surfaces (i.e., can be detected by contacting the target cell withan agent which specifically binds the protein). Also, the nucleic acidcan be a prophylactic agent useful in the prevention of disease.

A nucleic-acid category that is important to the present inventionencompasses polynucleotides that encode proteins that affectwound-healing. For example, the genes egf, tgf, kgf, hb-egf, pdgf igf,fgf-1, fgf-2, vegf, other growth factors and their receptors, play aconsiderable role in wound repair.

Another category of polynucleotides, coding for factors that modulate orcounteract inflammatory processes, also is significant for the presentinvention. Also relevant are genes that encode an anti-inflammatoryagent such as MSH, a cytokine such as IL-10, or a receptor antagonistthat diminishes the inflammatory response.

Suitable polynucleotides can code for an expression product that inducescell death or, alternatively, promotes cell survival, depending on thenucleic acid. These polynucleotides are useful not only for treatingtumorigenic and other abnormal cells but also for inducing apoptosis innormal cells. Accordingly, another notable nucleic-acid category for thepresent invention relates to polynucleotides that, upon expression,encode an anti-oncogenic protein or, upon transcription, yield ananti-oncogenic antisense oligonucleotide. In this context, the phrases“anti-oncogenic protein” and “anti-oncogenic antisense oligonucleotide”respectively denote a protein or an antisense oligonucleotide that, whenprovided to any region where cell death is desired, or the site of acancerous or precancerous lesion in a subject, prevents, inhibits,reverses abnormal and normal cellular growth at the site or inducesapoptosis of cells. Delivery of such a polynucleotide to cells, pursuantto the present invention, can inhibit cellular growth, differentiation,or migration to prevent movement or unwanted expansion of tissue at ornear the site of transfer. Illustrative of this anti-oncogenic categoryare polynucleotides that code for one of the known anti-oncogenicproteins. Such a polynucleotide would include, for example, a nucleotidesequence taken or derived from one or more of the following genes: abl,akt2, apc, bcl2-alpha, bcl2-beta, bcl3, bcl3, bcl-x, bad, bcr, brca1,brca2, cbl, ccnd1, cdk4, crk-II, csflr/fms, dbl, dcc, dpc4/smad4, e-cad,e2fl/rbap, egfr/erbb-1, elk1, elk3, eph, erg, ets1, ets2, fer, fgr/src2,fos, fps/fes, fra1, fra2, fyn, hck, hek, her2/erbb-2/neu, her3/erbb-3,her4/erbb-4, hras1, hst2, hstf1, ink4a, ink4b, int2/fgf3, jun, junb,fund, kip2, kit, kras2a, kras2b, ck, lyn, mas, max, mcc, met, mlh1, mos,msh2, msh3, msh6, myb, myba, mybb, myc, mycl1, mycn, nf1, nf2, nras,p53, pdgfb, pim1, pms1, pms2, ptc, pten, raft, rb1, rel, ret, ros1, ski,src1, tal1, tgfbr2, thra1, thrb, tiam1, trk, vav, vhl, waf1, wnt1, wnt2,wt1 and yes1. By the same token, oligonucleotides that inhibitexpression of one of these genes can be used as anti-oncogenic antisenseoligonucleotides.

Nucleic acids having modified internucleoside linkages also can be usedin composition according to the present invention. For example, nucleicacids can be employed that contain modified internucleoside linkageswhich exhibit increased nuclease stability. Such polynucleotidesinclude, for example, those that contain one or more phosphonate,phosphorothioate, phosphorodithioate, phosphoramidate methoxyethylphosphoramidate, formacetal, thioformacetal, diisopropylsilyl,acetamidate, carbamate, dimethylene-sulfide (—CH₂—S—CH₂—),dimethylene-sulfoxide (—CH₂—SO—CH₂—), dimethylenesulfone(—CH₂—SO₂—CH₂—), 2′-O-alkyl, and 2′-deoxy-2′-fluoro-phosphorothioateinternucleoside linkages.

For present purposes, a nucleic acid can be prepared or isolated by anyconventional means typically used to prepare or isolate nucleic acids.For example, DNA and RNA can be chemically synthesized usingcommercially available reagents and synthesizers by known methods. Forexample, see Gait, 1985, in: OLIGONUCLEOTIDE SYNTHESIS: A PRACTICALAPPROACH (IRL Press, Oxford, England). RNA molecules also can beproduced in high yield via in vitro transcription techniques, usingplasmids such as SP65, available from Promega Corporation (Madison,Wis.). The nucleic acid can be purified by any suitable means, and manysuch means are known. For example, the nucleic acid can be purified byreverse-phase or ion exchange HPLC, size exclusion chromatography, orgel electrophoresis. Of course, the skilled artisan will recognize thatthe method of purification will depend in part on the size of the DNA tobe purified. The nucleic acid also can be prepared via any of theinnumerable recombinant techniques that are known or that are developedhereafter.

A suitable nucleic acid can be engineered into a variety of known hostvector systems that provide for replication of the nucleic acid on ascale suitable for the preparation of an inventive composition. Vectorsystems can be viral or non-viral. Particular examples of viral vectorsystems include adenovirus, retrovirus, adeno-associated virus andherpes simplex virus. Preferably, an adenovirus vector is used. Anon-viral vector system includes a plasmid, a circular, double-strandedDNA molecule. Viral and nonviral vector systems can be designed, usingknown methods, to contain the elements necessary for directingtranscription, translation, or both, of the nucleic acid in a cell towhich is delivered. Methods which are known to the skilled artisan canbe used to construct expression constructs having the protein codingsequence operably linked with appropriate transcriptional/translationalcontrol signals. These methods include in vitro recombinant DNAtechniques and synthetic techniques. For instance, see Sambrook et al.,1989, MOLECULAR CLONING: A LABORATORY MANUAL (Cold Spring HarborLaboratory, New York), and Ausubel et al., 1997, CURRENT PROTOCOLS INMOLECULAR BIOLOGY (John Wiley & Sons, New York).

A nucleic acid encoding one or more proteins of interest can beoperatively associated with a variety of different promoter/regulatorsequences. The promoter/regulator sequences can include a constitutiveor inducible promoter, and can be used under the appropriate conditionsto direct high level or regulated expression of the gene of interest.Particular examples of promoter/regulatory regions that can be usedinclude the cytomegalovirus (CMV) promoter/regulatory region and thepromoter/regulatory regions associated with the SV40 early genes or theSV40 late genes. Preferably, the human CMV promoter is used, butsubstantially any promoter/regulatory region directing high level orregulated expression of the gene of interest can be used.

It also is within the scope of the present invention that the employednucleic acid contains a plurality of protein-coding regions, combined ona single genetic construct under control of one or more promoters. Thetwo or more protein-coding regions can be under the transcriptionalcontrol of a single promoter, and the transcript of the nucleic acid cancomprise one or more internal ribosome entry sites interposed betweenthe protein-coding regions. Thus, a myriad of different genes andgenetic constructs can be utilized.

In certain embodiments of the composition, the biomaterial is a memberselected from the group consisting of an antibody, a viral vector, agrowth factor, a bioactive polypeptide, a polynucleotide coding for thebio active polypeptide, a cell regulatory small molecule, a peptide, aprotein, an oligonucleotide, a gene therapy agent, a gene transfectionvector, a receptor, a cell, a drug, a drug delivering agent, nitricoxide, an antimicrobial agent, an antibiotic, an antimitotic, dimethylsulfoxide, an antisecretory agent, an anti-cancer chemotherapeuticagent, steroidal and non-steroidal anti-inflammatories, hormones, anextracellular matrix, a free radical scavenger, an iron chelator, anantioxidant, an imaging agent, and a radiotherapeutic agent. Preferably,the biomaterial is at least one of an anti-knob antibody, an adenovirus,a D1 domain of the Coxsackie-adenovirus receptor (CAR D1), insulin, anangiogenic peptide, an antiangiogenic peptide, avidin, biotin, IgG,protein A, transferrin, and a receptor for transferrin. A combination ofmultiple types of biomaterials bound to each other by affinity is alsocontemplated. For example, an adenovirus bound to a D1 domain of theCoxsackie-adenovirus receptor (CAR D1) or to a specific antibody can beattached to the matrix via the water-soluble photo-activatable polymer.

Antibodies specific for non-viral vectors or nucleic acid may requireuse of a transfection agent to enhance administration of nucleic acid.The transfection agent is a cationic macromolecule that is positivelycharged, comprises two or more art-recognized modular units (e.g., aminoacid residues, fatty acid moieties, or polymer repeating units), and ispreferably capable of forming supermolecular structures (e.g.,aggregates, liposomes or micelles) at high concentration in aqueoussolution or suspension. Among the types of cationic macromolecules thatcan be used are cationic lipid and polycationic polypeptides.

The amount of the transfection agent to be used when transfecting cellscan be calculated based on nucleic acid content of the composition. Thecapacity of the medium comprising or containing the transfection agentcan also affect the amount of transfection agent to be used. When theantibody of the transfection agent is immobilized on a matrix, theamount of cationic macromolecule and DNA that can be complexed with theantibody can be limited by the physical requirements of the metalsupport. For example, rigidity, flexibility and chemical reactivity mayinfluence the amount of transfection agent used. Such vectors haveincluded retroviral, adenovirus, adeno-associated viral vectors andherpes viral vectors. Cells can be infected with viral vectors by knownmethods.

Further provided is a method of making the composition of the invention,the method comprising providing the matrix having at least one carbon;providing an aqueous solution of the water-soluble photo-activatablepolymer having the photo-activatable group and the reactive group; andphoto-activating the photo-activatable group by irradiation tocovalently attach the water-soluble polymer via the photo-activatablegroup to the matrix and thereby forming the monomolecular layer of thecomposition on the matrix. Example 3 describes the conditions of themethod using PAA-BzPh and PDT-BzPh as non-limiting examples.

In certain embodiments of the method of making the composition of theinvention, the irradiation is performed at a wavelength from about 190to about 900 nm. Preferably, the irradiation is performed at awavelength of 280 to 360 nm.

Additionally, certain embodiments of the method further compriseproviding a biomaterial having a plurality of active groups and reactingthe plurality of active groups with the water-soluble photo-activatablepolymer to covalently attach the biomaterial to the matrix. Conditionsof such reaction should not be damaging for the biomaterial to beattached. These conditions should involve using buffers in thephysiologic range (pH 7.35-7.45) and osmotic strength, and temperatureconditions between 25C and 37C, but not higher or lower.

Photochemical Modification of Micro- and Nanoparticles

The water-soluble photo-activatable polymers of the invention obtainedas described above can be bound to the surface of pre-formed micro- ornanoparticles (MP and NP, respectively) to form modified particlescapable of reacting with the biomaterial.

Advantageously, in the preparation of biodegradable andnon-biodegradable polymeric micro- and nanoparticles, the water-solublephoto-activatable polymer of the invention (e.g., BzPh-PDT) not onlymakes the surface reactive towards biomolecules containing suitablereactive groups (e.g., the thiol-containing biomolecules), but alsoprevents the flocculation of particles and thus stabilizes thesuspension.

Particles modified with water-soluble photo-activatable polymers of theinvention can be delivered via various delivery routes to an organism.For example, injectable nanoparticles can be used to either provide anintravenous means of sustained delivery of proteins and peptides, or ifinjected into a specific site such as a tumor, or the myocardium, canprovide sustained local presence of therapeutic peptides and proteins.

Particles size and composition can be tailored to a specificapplication. Particles can be prepared by one of the existing methods(see Couvreur P et al., Nanoparticles: preparation and characterization.In: Benita S, Editor, Microencapsulation. Methods and industrialapplications. vol. 73. New York: Marcel Dekker, 1996. pp. 183-211; KumarMNR. Nano- and microparticles as controlled drug delivery devices. JPharm Pharmaceut Sci 2000; 3:234-58).

Most widely used are methods including emulsification-polymerization andpolymer precipitation techniques. The former may be accomplished by insitu polymerization of monomers either in aqueous solution or emulsifiedin aqueous phase (namely, emulsification-polymerization). Alternatively,methods exploiting pre-formed biocompatible polymers, usually ofpolyester and polyanhydride families, can be used to form particles bypolymer precipitation methods. The most popular methods areemulsification-solvent evaporation, emulsification-diffusion andnanoprecipitation methods (see Quintanar-Guerrero et al., Preparationtechniques and mechanisms of formation of biodegradable nanoparticlesfrom preformed polymers. Drug Dev Ind Pharm 1998; 24:1113-28). Thelatter methods are based on emulsifying an organic solution of a polymerwith or without drug in an aqueous phase in presence of a stabilizersubstance (e.g., Poloxamer 188, polyvinyl alcohol, etc.) achieved eitherby external energy input or through spontaneous diffusion ofwater-miscible solvents with subsequent solvent elimination to formsolid particle dispersion.

While both emulsification-polymerization and polymer precipitation areapplicable for preparing matrix-type particles (spheres), some of thesemethods with appropriate modifications can be used for producingcore-shell type vesicles (capsules). The drug substance can either beencapsulated (dissolved or dispersed in the polymeric matrix of a sphereor dissolved in the liquid core of a capsule) or adsorbed/chemicallybound to the particle surface. The latter approach where a substance isattached to the surface of a preformed particle has the advantage ofavoiding harsh conditions (extreme pH, exposure to organic solvents orelevated temperatures) employed for the particle formulation. Covalentassociation of the drug with the particle surface employingbiodegradable chemical bonds (e.g., by disulfide linking) provides analternative that achieves both controlled and site-specific release ofthe drug. Further, affinity based association of one type of biomaterialwith another type of biomaterial, which it turn is covalently found tothe water-soluble photo-activatable polymer of the invention coupled tonanoparticles provides a novel delivery option for biomaterial based onthe release of affinity bonds (see FIG. 5).

Attachment of biomaterial to NP has been demonstrated using D1, IgG andadenovirus. It should be understood that these embodiments arenon-limiting examples.

Adenovirus (AdV or Ad were used in this disclosure interchangeably) is apromising gene vector for therapeutic applications. However, its use iscompromised, since AdV-mediated gene transfer is suboptimal in celltypes deficient in Coxsackie-AdV receptor (CAR). Inventors havediscovered that adenovirus vector (AdV) delivery and transgeneexpression at levels equivalent to or greater than with CAR-processingcould be achieved through a receptor-independent mechanism by affinitytethering AdV to the surface of biodegradable nanoparticles (NP).

In certain embodiments of the composition, the viral vector isadenoviral vector (AdV) encoding a desired protein such as, for example,GFP or inducible NO synthase (iNOS).

NP (350-450 nm) were prepared by a modified emulsification-solventevaporation method and surface-modified by an anionic thiol- andphotoreactive poly(allylamine) derivative by a brief exposure to UVlight. The NP were coated with a thiolated D1 domain of CAR ornon-immune IgG as a control, and associated with GFP- or inducible NOsynthase (iNOS)-encoding AdV. The uptake of BODIPY 564/570-labeled NPand GFP expression were assayed fluorimetrically. Cell growth inhibitionwas determined in A10 cells two days post transduction using theAlamarBlue assay. The gene expression in A10 cells treated with AdV-NPcomposites was found to be dependent on the D1-modified NP amount andequaled several times that of free AdV treated cells, while no increasein efficacy was shown for the AdV applied with IgG-modified NP despitean equally effective uptake of the two NP types. Exposure to knobprotein resulted in a complete inhibition of gene expression mediated byfree AdV, whereas no change in the transduction efficacy was observedfor AdV-NP. Composites formulated with iNOS AdV effectively inhibitedA10 cell growth (e.g., 58% inhibition), whereas iNOS AdV alone or inpresence of IgG-coated NP had a substantially lower effect on the cellproliferation (e.g., 0% and 22% inhibition, respectively). Thisinhibitory effect correlated well with the gene expression measuredusing GFP AdV reporter. These results demonstrate that AdV-NP compositestaken up via a receptor-independent pathway can substantially increasegene transfer in vitro. This strategy is therefore advantageous fortransduction of CAR-deficient cells. The potent smooth muscle cellgrowth inhibitory effect achieved with iNOS AdV-NP composites makes thisdelivery system a promising candidate for gene therapy of proliferativedisorders (e.g., for restenosis, cancer treatment, vasodilation andpulmonary hypertension). Also provided is a method for delivery of abiomaterial to a cell or an organism, the process comprising (1)providing the composition of the invention as a monomolecular layer anda matrix having at least one carbon, wherein the monomolecular layer iscovalently attached to the matrix by a covalent bond between thephoto-activatable group and the at least one carbon, (2) providing abiomaterial having a plurality of active groups, wherein the biomaterialis covalently attached to the monomolecular layer by covalent bondingbetween the active groups and the reactive groups; and (3) administeringthe matrix to the cell or an organism.

In vivo studies examined the levels and regional differences in Adluciferase reporter (_(LUC)Ad) gene expression comparing NP-Ad complexesand free Ad administered subdermally in rats using quantitativebiophotonic imaging.

Amounts of the biomaterial may vary depending on the purpose ofdelivery, e.g., prophylactic, diagnostic, therapeutic, etc. and on thenature of the biomaterial involved.

In certain embodiments, the biomaterial delivered by this method is thebiomaterial is at least one of a protein, a D1 domain of theCoxsackie-adenovirus receptor, an adenovirus, or an antibodyspecifically bound to a nucleic acid.

These investigations have demonstrated a number of major findingsincluding: 1) the synthesis of a multifunctional thiol- andphotoreactive polymer (PBPC/PBMC) for chemically activating the surfaceof nanoparticles, 2) the formulation of surface activated (viaPBPC/PBMC) biodegradable NP that mediate viral vector delivery throughNP-surface tethering with covalently attached vector binding proteins,and 3) demonstrating that these NP-Ad complexes achieve gene expressionlevels both in vitro and in vivo that exceed those with equivalentamounts of free Ad. Other groups have not reported the synthesis andfunctionality of photoreactive polymers comparable to PBPC, which can infact react following ultraviolet light exposure with virtually any C—Hbond, thus making this reagent a particularly potent tool for creatingsurface modifications for a wide variety of synthetic and biopolymers.PLA as a substrate is abundant with C—H bonds capable of reaction withthe excited aromatic ketone functions of PBPC, which is anotherimportant advantage of the present approach as opposed to a recentlydescribed end-group modification of NP-forming PLA (Nobs et al., 2003,2004) that is limited by the number of exposed carboxylic functions.

The vector tethering strategy reported herein has previously beeninvestigated by our group for gene delivery from the surfaces ofcollagen coated stents using anti-knob antibodies (Klugherz et al.,2002). However, the present NP studies are the first demonstration ofNP-Ad delivery with D1 as a tethering agent. The receptor-independentmechanism for Ad-NP cell entry is another important unique feature ofthis formulation, enabling the transduction of cells with relatively lowto absent levels of CAR.

Several of the individual components of the present formulations havebeen investigated by others with results that support the findings ofour studies. Surface tethering of viral vectors to nondegradableparticles has been investigated and varying degrees of gene expressionenhancement were observed in these studies following delivery of Ad in aform of conjugates to nondegradable nano- and microbeads in vitro and invivo (Pandori et al., 2002a; Qiang et al., 2004; Pandori and Sano,2005). However, an increased gene transfer by Ad immobilized on thesurface of biodegradable NP using an Ad-specific binding protein has notbeen described. Furthermore, while the potentiating effect ofassociation with solid particles has been demonstrated for differenttypes of viral vectors, including lentivirus (Haim et al., 2005),retrovirus (Hughes et al., 2001) and adeno associated virus (Mah et al.,2002), the mechanism of the enhancement has not been studied insufficient detail, and its correlation with the nature of the virusbinding to the carrier particles and their cell entry has not beenclearly delineated in these prior investigations.

The D1 protein used in this study for Ad immobilization is anextracellular immunoglobulin-like domain of CAR possessing a highbinding affinity to the adenoviral fiber knob domain (K_(d) in the orderof 20-25 nM) (Lortat-Jacob et al., 2001). The approach employing D1 asan Ad-specific affinity ligand allows for immobilization of the viralvector on the preformed NP in one step without need for prior chemicalmodification of the virus that may potentially result in a substantialloss of vector infectivity (Pandori et al., 2002b). The modification ofthe NP with D1 was accompanied by some increase in the particle sizethat can be attributed to the layer of the surface-bound protein,whereas the colloidal stability of the formulation was not adverselyaffected. Furthermore, NP coated with D1 remained stable in suspensionupon binding Ad in contrast to the _(AK)NP. The colloidal stabilitycorrelated with the gene transfer efficacies of the respectiveformulations favoring the submicronial-sized _(D1)NP-Ad complexesexhibiting high cell entry capacity, and is also of importance in termsof safety of their use in vivo.

NP-Ad were efficiently taken up by cells and exhibited sustainedelimination kinetics with more than half of the initial particle loadresident in the cells interior after 72 hr (FIG. 3). Despite the highintracellular levels achieved within 2 hr of exposure, the complexeselicited no cell toxic effects as evidenced by cell growth that wasunaffected by _(D1)NP-_(NULL)Ad at any dose in the studied range (FIG. 4F). The moderate cell toxicity caused by the GFP-encoding complexes(FIG. 4 E) should therefore be attributed to the GFP gene product thatis known to be toxic when expressed at high levels (Liu et al., 1999).The NP-Ad internalization occurred through a CAR-independent mechanism,which is in compliance with relative increase in transduction that wasmost significant in the H5V cells that are completely devoid of CARexpression, and is also supported by data showing the recovery of viralgene expression inhibited by knob protein achieved by vector associationwith _(D1)NP. This is in agreement with the results recently reported byPandori et al. (Pandori and Sano, 2005) who observed equal infectivityof Ad immobilized on non-biodegradable silica microbeads in the presenceor absence of anti-CAR antibody used to block the CAR-mediated cellentry. Smooth muscle and endothelial cells are known to expressrelatively low levels of CAR (Wickham et al., 1996; Wickham et al.,1997) necessitating the use of high titres of Ad in order to achievesufficiently high levels of transgene expression in vascular cells invitro and in vivo (Baker, 2004). The potent CAR-independent cellularuptake of the NP-Ad complexes resulting in a strongly enhanced geneexpression by these cell types observed in our study may therefore be ofrelevance for the development of effective Ad-based therapies forcardiovascular disease.

The enhancement of the gene transfer efficacy was specific to NP thatwere surface-modified with the binding protein enabling Adimmobilization, and was directly dependent on the doses of Ad and NP(FIGS. 11A-B), which is in accord with the uptake dependingnear-linearly on the NP amount used to form the complexes (FIGS. 9A-B).However, a comparable degree of internalization was observed for control_(nIgG)NP and these NP exhibited no infectivity enhancement (FIGS. 7A-F)apparently due to their inability to promote viral cell entry requiringa sufficiently strong NP-Ad association. The high intracellular levelsof the complexes observed in cell culture with resultant increase ingene expression appear to be due to a combined effect of thevector-binding particles concentration on the cell surface (Luo andSaltzman, 2000; Pandori and Sano, 2005), and the rapid kinetics of theCAR-uncoupled internalization. The potentiating effect of the Adimmobilization was most pronounced at low doses of Ad reaching transgenelevels in A10 cells up to 45-fold higher compared to the equivalent doseof free Ad. The earlier onset of expression mediated by the complexesresulted in a notably higher gain in the transgenic product levels inA10 and BOEC 24 hr post treatment than at later timepoints. However, thedifference in the GFP expression level between the complexes andcontrol-treated cells remained significant over 7 days.

The mechanism of the virus dissociation from the carrier particle maypotentially have an effect on the efficacy and kinetics of geneexpression in vitro and in vivo. The substitution of the reducibledisulfide bond between the NP and the Ad binding protein for anon-degradable thio ether linkage (_(PDT-D1)NP vs. _(MI-D1)NP) resultedin similar patterns of the reporter expression for the two formulations(FIGS. 10 B and 10 C), which given comparable rates of their cellularuptake suggests the same fate of the internalized viral vector in bothcases. Therefore, it seems plausible that the dissociation of the D1-Adbond accounts for the release of the virus from the complex. Whereas astrong binding between NP and Ad is obviously important, theincorporation of a degradable bond apparently is not a prerequisite foreffective transduction achievable by this method.

Growth inhibition of A10 cells using an inducible NO synthase (iNOS)encoding vector was chosen to examine the ability of the _(D1)NP-Adcomplexes to exert a therapeutically relevant effect. _(iNOS)Ad deliverywith NP has not been demonstrated prior to the present studies. However,_(iNOS)Ad administered as suspensions of free vector has been shown tohave pleiotropic effects on a number of mechanistic targets in thearterial wall including inhibition of arterial smooth muscleproliferation, inhibition of platelet activation, and enhanced arterialwall relaxation. Thus, NP-_(iNOS)Ad are of potential importance fornovel therapies for vascular disease in view of the broad therapeuticimpact of iNOS. A profound inhibition of aortic smooth muscle cellgrowth in culture was observed following treatment by the_(D1)NP-_(iNOS)Ad complexes, whereas no inhibitory effect was exhibitedby complexes formulated with null Ad or _(iNOS)Ad applied in thepresence of control _(nIgG)NP (FIGS. 4A-F). GFP reporter expression andcell growth inhibition by the two respective types of NP-Ad revealed asimilar pattern suggesting that the expression of iNOS is the maindeterminant of the aortic smooth muscle cell growth inhibition in vitro,and confirming the utility of complexes formulated with areporter-encoding Ad as a useful model.

The results of the examples described below need to be viewed in thecontext of several limitations of this work. For example, the long termbiodegradation of the PLA core of the NP was not investigated; the halftime of PLA degradation ranges from several weeks to several months(Laurencin and Elgendy, 1994; Perrin and English, 1997). Thus, whilethis parameter would not affect the rapid processing of the NP-Adcomplexes observed in the present results, the fate of NP over timewould be of concern and will be addressed in longer term studies. Thedissociation constants for Ad bound to NP were not determined in thesestudies. Prior investigations indicate the K_(d) in solution for theantibody used is 0.31 nM (Nyanguile et al., 2003), and the K_(d) for D1is 20-25 nM (Lortat-Jacob et al., 2001). However, when these Ad-bindingproteins are immobilized onto surfaces the K_(d) may differ, and thishas been observed for D1 (Lortat-Jacob et al., 2001). This parameter isalso of potential importance in terms of the long term fate of free Ad,and is of special importance since the results of the present study showthat Ad-binding agent affinity and not covalent attachment of thebinding agent to the NP surface determines Ad release. Furthermore,intracellular trafficking of NP and Ad before and after dissociationinvolves complex events, and should also be the subject for futureinvestigations. The in vivo experiment reported herein demonstratedsignificantly greater early levels of gene expression with NP-Ad versusfree Ad; however 5 day expression levels were comparable. These resultscan best be explained by the fate of NP-Ad following subcutaneousinjection when there are principally interactions of NP-Ad withmobilized white blood cells that are transient and likely traffic out ofthe injection region by the five day time point. Nevertheless, an invivo efficacy experiment involving a therapeutic endpoint wouldconstitute a more definitive comparison of NP-Ad and free Ad.

NP-Ad complexes formed with PBPC-activated PLA NP, that weresurface-modified with vector binding proteins can deliver Ad to cells ina CAR independent manner, resulting in higher levels of transgeneexpression both in vitro and in vivo than achieved with administrationof comparable amounts of free Ad. These discoveries have importantimplications for the safe and efficacious use of adenoviral vectors ingene therapy.

Delivery of Biomaterial Via Magnetic Nanoparticle

An additional degree of site-specificity may be achieved by renderingthe particles (with and without biomaterial) responsive to magneticfield (e.g., superparamagnetic) by inclusion a magnetic field-responsiveagent (e.g., magnetite/maghemite nanocrystals) in the polymeric matrix(e.g., Ito R, Machida Y, Sannan T, Nagai TU-hwscsaBTW-B-Gddaaca.Magnetic granules: a novel system for specific drug delivery toesophageal mucosa in oral administration. Int J Pharm 1990; 61:109-117).This modification allows for concentrating the particles at their targettissue using magnetic field, thereby increasing their therapeuticefficacy and minimizing the formulation toxicity.

A magnetic field-responsive agent as used herein is a paramagnetic,superparamagnetic, or ferromagnetic substance capable of moving underinfluence of a magnetic force. Superparamagnetic material are preferredmaterials. In certain embodiments, the magnetic field-responsive agentis a member selected from the group consisting of iron, cobalt ornickel, alloys thereof, oxides thereof and mixed oxides/hydroxides ofFe(II) and/or Fe(III) with at least one of Co(II), Mn(II), Cu(II),Ni(II), Cr(III), Gd(III), Dy(III), and Sm(III). Preferably, the magneticfield-responsive agent is at least one of Fe3O4, gamma-Fe2O3, or amixture thereof. Preferably, the magnetic field-responsive agent is ironoxide in a shape of nanocrystals.

The magnetic field-responsive agent can be prepared by methods known inthe art in various shapes and sizes (see Hyeon T., Chemical Synthesis ofMagnetic Nanoparticles. The Royal Society of Chemistry 2003, Chem.Commun., 2003, 927-934). In certain embodiments, iron oxide nanocrystalswere obtained by precipitation of mixed iron chlorides in the presenceof a base in aqueous medium (see Khalafalla S E. Magnetic fluids,Chemtech 1975, September: 540-547).

FIG. 20 is a schematic representation of delivering magneticnanoparticles carrying GFP encoding adenovirus to magnetic fieldgradients produced by 316L steel. The therapy (green glow) is deliveredonly to the cells growing on top of the 316L stent wires, in order toindicate the nature of local delivery method. The magnetic field acrossthe mesh causes the development of high field gradients between the meshwires which attracts and binds magnetically responsive nanoparticles.The conceptual model (see FIG. 20) for delivery of magneticnanoparticles may be described mathematically with analyticalexpressions that involve several assumptions detailed as follows.

In obtaining the force required for delivery of magnetic nanoparticles,it is reasonable to neglect the field gradients throughout thewire-metallic meshwork from all but the directly adjacent wires, sincethe field gradient decays inversely with the cube of distance away fromthe wire center. Thus, if the biomolecular-nanoparticle complexes arelocated within a few wire diameters from the struts, it is reasonable toapproximate the wire as an infinitely long cylinder. For such a case,the wire's magnetic potential (_(wire) can be approximated by thefollowing expression, assuming the wire is magnetized orthogonal to itslong axis:

$\begin{matrix}{{\phi_{wire}\left( {\overset{\rightarrow}{r},\theta} \right)} = {\frac{\lambda_{wire}}{2\; \pi}\frac{\cos (\theta)}{\overset{\rightarrow}{r}}}} & (1)\end{matrix}$

where {right arrow over (r)} is the position vector of the fieldmeasurement point, θ is the angular elevation of the position vectorwith respect to the wire's magnetization, and λ_(wire) is theexperimentally determined magnetic property of an infinitely thin wirehaving units of A m, denoting an effective magnetic moment per unitlength. The wire magnetization may in general be a constant or anonlinear function of the external field. The magnetic field produced bythe wire can be calculated by taking the negative gradient of themagnetic scalar potential as follows:

{right arrow over (H)} _(wire) =−∇φ.

Superparamagnetic nanoparticles formulated primarily from ferrite areused in a limited number biomedical applications, because they lackremnant magnetization and therefore do not tend to form irreversibleaggregates. Ferrites are ferromagnetic ceramic materials, compounds ofiron (e.g., iron (III) oxide), boron and barium or strontium ormolybdenum. Ferrites have a high magnetic permeability, which allowsthem to store stronger magnetic fields than iron, and are known asceramic magnets.

Magnetization behavior of such nanoparticles typically follows anonlinear relationship with the external field, described by thewell-known Langevin's function. In order to achieve an analyticalsolution related to the nanoparticles of the invention, it is proposedto model the nanoparticle behavior using a hard saturation model,consisting of a linear regime in low fields followed by a constantsaturation region in high fields. In the low field regime, the magneticmoment (m) of the nanoparticle is given in expression (2), which is thewell-known result for the magnetic moment of a homogeneous isotropicsphere magnetized by a uniform magnetic field (see Yellen B B, Forbes ZG, Halverson D S, Fridman G, Barbee K A, Chorny M, Levy R J, Friedman G.Targeted drug delivery to magnetic implants for therapeuticapplications. Journal of Magnetism and Magnetic Materials, 293:647-654,2005)

$\begin{matrix}{\overset{\rightarrow}{m} = {\frac{3\; \chi}{\chi + 3}V\overset{\rightarrow}{H}}} & (2)\end{matrix}$

where V is the volume of the spherical particle, x is the magneticsusceptibility, and H is the external field at the location of theparticle. Although in this work, the field is designed to beintentionally inhomogeneous for attracting the particles, the field doesnot vary rapidly across the substantially smaller magnetic nanoparticlediameter, and thus the dipole moment is still expected to be a goodapproximation. Thus, the total magnetic field at the center of thenanoparticle is due to the field created by the wires of the stent, thefield due to other nearby particles, and any externally applied field.If one considers weakly concentrated nanoparticle solutions whereparticles are separated from one another by at least 10 nanoparticlediameters, then particle-particle interactions are negligible and onlydirect particle-wire interactions need be taken into account.

The expression (3) defines magnetic force (F_(mag)) on a particle asfollows:

$\begin{matrix}{{\overset{->}{F}}_{mag} = {{{\mu_{0}\left( {\overset{->}{m} \cdot \nabla} \right)}\overset{->}{H}} \approx {\mu_{0}\frac{3\; \chi}{\chi + 3}{V\left( {\overset{->}{H} \cdot {\nabla\overset{->}{H}}} \right)}}}} & (3)\end{matrix}$

where μ₀ is the magnetic permeability of free space. When modelinghydrodynamic systems, the typical approach is to ignore particle inertia(any particle acceleration happens over time periods that are tinycompared to typical time of particle movement). Thus, the particlevelocity can be obtained by equating the magnetic force in (4) to theStoke's drag force, as given by:

{right arrow over (F)} _(drag)=3πdη({right arrow over (ν)} _(f) −{rightarrow over (ν)} _(p))  (4)

where η is the fluid viscosity, d is the particle diameter, {right arrowover (ν)}_(f) and {right arrow over (ν)}_(p) are the fluid and particlevelocities, respectively. In order to obtain a rough estimate for thedynamics of particle capture, consider the case of a particle initiallylocated at a distance D from the wire's center. Assuming the optimalscenario of the particle being located in a stationary fluid, and thewire and nanoparticle magnetizations are aligned collinearly with thevector connecting their two centers, it is possible to obtain ananalytical expression for the time interval t it would take to capture ananoparticle onto the wire's surface of radius R, which is given byexpression (5):

$\begin{matrix}{D = {\left\lbrack {{H_{0}\frac{2\mu_{0}d^{2}{\lambda \left( H_{0} \right)}t}{3{\pi\eta}}\frac{\chi}{\left( {\chi + 3} \right)}} - R^{4}} \right\rbrack^{1/4} - R}} & (5)\end{matrix}$

wherein H_(o) is initial magnetic field.

In this analysis, it was assumed that the external magnetic fielddominates the wire's field, and hence the wire's contribution tomagnetizing the particle was ignored in the above expression. For weaklymagnetic materials, such as 316L, this assumption is accurate even whenfields of only 10 Oerstad are applied. From (5) it is clear that themost effective control parameters for tuning nanoparticle delivery arethe nanoparticle diameter, the external field, the wire magnetization,and the time interval. All of these control parameters have beeninvestigated, and the results from theoretical simulations are shown tobe in good agreement with experimental investigations.

Inventors have discovered that magnetic particles of the invention canbe delivered to a magnetic implanted device (e.g., a stent) byadministration to a body (e.g., an intravenous injection). In that,certain parameters can be manipulated to control the nanoparticledelivery, e.g., the nanoparticle diameter, the external field, theimplanted device surface magnetization, and the time interval it wouldtake to capture the nanoparticle onto the surface.

The term “bioactive magnetic particle” as used herein denotes a magneticparticle associated with biomaterial, which is covalently attached to amonomolecular layer formed on the surface of the particle. It should beunderstood that the term “bioactive magnetic particle” is not limited toparticles delivering biomaterial. Any molecules capable of attachment toparticles as described above, such as, for example, imaging agent can beused also.

Further provided is method for delivery of a biomaterial to a cell or atissue, the method comprising: (a) providing a bioactive magneticparticle comprising (1) a matrix having at least one carbon, wherein thematrix is in a shape of a particle having a diameter of about 5 nm toabout 10 microns, (2) the water-soluble photo-activatable polymer as amonomolecular layer covalently attached to the matrix by a covalent bondbetween the photo-activatable group and the at least one carbon; (3) thebiomaterial having a plurality of active groups, wherein the biomaterialis covalently attached to the monomolecular layer by covalent bondingbetween the active groups and the reactive groups; and (4) magneticfield-responsive agent associated with the particle; (b) providing animplant comprising a magnetic surface to the cell or the tissue; (c)administering the bio active magnetic particle to the cell or thetissue; and (d) capturing the bioactive magnetic particle onto themagnetic surface. Administering the bioactive magnetic particle can bedone, for example, by injection.

In certain embodiments, delivery of the biomaterial to a cell or atissue can be manipulated by selecting at least one of a particlediameter, proximity of an external magnetic field, a degree of surfacemagnetization of the implant, and a time interval for capturing theparticle onto the magnetic surface.

Theoretical Simulations

The magnetic properties of all the materials were measured with analternating gradient magnometer (AGM) (Princeton MeasurementsCorporation, Princeton, N.J.). Hysteresis curves were generated for thenanoparticles, the bare 316L stainless steel wire, and the 316Lstainless steel wire coated with Nickel/Cobalt alloy. From thehysteresis curves, the initial susceptibility of the 380 nm diametermagnetic nanoparticles was found to be in the range of 0.1˜1.0, which isconsistent with the properties of commercially available materials(Dynal Biotech, NY) composed of approximately 10% magnetite by volume.The magnetic moments of the stent materials were also measured, andtheir behavior is expressed in terms of moment per unit length tomaintain consistency with the wire magnetization parameters used insimulations. The magnetic moment per unit length of the Palmaz-Shatzstent wire was found to relate linearly with the externally appliedfield H₀ in the range of 0 to 5000 Oerstads according to:λ_(316L)=9.2·10⁻¹¹ m²·H₀+3.3·10⁻⁶ A·m. The magnetic properties of asimilar segment coated with a 20 μm thick layer of NiCo alloy was shownto relate linearly with the externally applied field between 0 and 700Oerstaeds as: λ_(316L+NiCo)=5.8·10⁻⁸ m²·H₀+1.2·10⁻⁴ A·m, which is two tothree orders of magnitude stronger than bare 316L steel.

Theoretical simulations were carried out using MATHCAD software todemonstrate the feasibility of capturing magnetic nanoparticles withexisting unmodified and modified 316L stents implanted in plastic tubeswith 3.2 mm inner diameter. A series of simulations were carried out todetermine the effect of various control parameters as well as generalscaling principles. In all simulations, each nanoparticle was assumed tohave initial susceptibility of 0.5, and the particle was assumed to movetowards a stent wire of 150 μm diameter through a fluid with a viscosityof 0.03 poise, which is consistent with that of blood. The theoreticaldesign was chosen to simulate a catheter-based nanoparticle deliverydevice, such as the double balloon catheters, which have the capabilityof arresting blood flow in certain regions for time frames on the orderof 60 seconds. The capture percentage is equal to the ratio of thecapture distance in expression (5) with respect to the radius of theplastic tube. The physical meaning of this ratio suggests that if thewire can capture a particle located at a distance of one tube radius,then all particles in suspension can be captured.

The capture percentage was first determined as a function of the appliedfield. As shown in FIG. 16A, roughly 2-3% of the particles can becaptured within the first 60 seconds on bare 316L stainless steel,whereas 20-30% of the particles can be captured in a similar interval onthe Nickel/Cobalt modified 316L steel, as shown in FIG. 16B. The insetwithin each figure is provided to give an indication of the capturedynamics occurring over the first 100 milliseconds, showing thepotential of magnetic nanoparticles to be captured in normalphysiological flow conditions. Although this model estimate ignoresseveral effects, such as the presence of other stent wires and the edgesof the stent itself, as well as lift forces and other hydrodynamiceffects induced by the boundary of the arteries, it is believed that anorder of magnitude estimate can still be obtained using static captureanalysis. Using the simple estimates of 1 cm/s fluid flow rate near thearterial wall and 100 μm diameter wire, it is possible to arrive at atime constant of 10 milliseconds, which is the time interval for which aparticle in transit will cross one of the strut wires during blood flow.This analysis indicates that the particle must be captured within thefirst 10 milliseconds if it has any chance of being delivered by simpleintravenous injection. Simulations suggest that bare 316L wire hasvirtually no chance of capturing nanoparticles within the first pass(<0.01%). Even if “stealth” nanoparticles, which are surface modified(with polyethyleneglycol for example) to prolong their persistence inthe circulation, are employed to evade capture by macrophages and allowparticles to survive inside the cardiovascular system for 100 passesthrough the coronary artery, the total capture is not expected to exceed1%. The Nickel/Cobalt coated 316L steel, by contrast, has a chance ofcapturing a fraction of particles in each pass (roughly 0.5% per pass).Given a sufficient number of passes this analysis indicates that a largefraction of nanoparticles can be localized on such strongly magneticstents.

The modeled capture percentage was also analyzed as a function ofnanoparticle diameter. Due to general scaling principles, there is anadvantage to using larger particles since the magnetic force increaseswith the cube of particle diameter whereas the drag force increases onlylinearly with particle diameter. Practically speaking, biologicalapplications would be limited to using particles that can fit throughthe capillary beds in the cardiovascular system, restricting the maximumsize of the particles to around 1000 nm. The capture dynamics ofdifferent nanoparticle diameters were simulated and the results areprovided in FIG. 3 c. In these simulations, the particle's moments wereassumed to be saturated by an externally applied 1000 Oerstad field. Asexpected, over the course of 60 seconds the 1000 nm particles can becaptured at higher levels (up to 5%) than the 400 nm particles (up to2%) used in most of the present experiments.

Experimental Results—In Vitro

Theoretical predictions simulating local delivery of 380 nm sizedpolylactide-based magnetic nanoparticles to 316L stents were testedexperimentally in simple stop-flow conditions. Stents were deployed byballoon catheter in 3.2 mm diameter plastic tubes filled with a buffersimulating blood pH and viscosity. Magnetic nanoparticles were injectedinto the tube nearby the stent, and then exposed to uniform 1000 Oerstadfields for 60 seconds. As a control, some stents were exposed tomagnetic nanoparticles for similar time duration without exposure to anexternal field. Afterwards, the stents were removed, cut open, pressedflat en face on a glass slide, and imaged by fluorescent microscopy.Examples of the fluorescent images obtained from individual wires of316L stents are shown in FIG. 17A-F. Figs A-B show representative photostaken from unmodified Palmaz-Shatz stents exposed to magneticnanoparticles without application of external magnetic field, whereasFIG. 17 C-D show the magnetic nanoparticle accumulation on similarstents after exposure to 1000 Oerstad magnetic field. Quantitativefluorescent analysis revealed that the external field increased particlecapture by roughly 10 times over the stents that were not exposed to anexternal field. FIG. 17 E-F shows the attraction of magneticnanoparticles to the Palmaz-Shatz stents coated with Nickel/Cobalt alloyin 1000 Oerstad field. The relative fluorescence over commensurate areaswas more than 25 times that of the unmodified Palmaz-Shatz stent undercomparable conditions.

The percentage of particle capture was evaluated by semi-quantitativefluorescence analysis using a known concentration from the stocksolution as a reference. Results suggest that the unmodified 316L steelstent captured roughly 1% (+/−0.3%) of the injected particles, whereasthe Nickel/Cobalt coated stent captured roughly 25% (+/−5%) of theinjected particles. Estimations were based on a 2.5 cm² stent surfacearea, leading to numbers which agree not only qualitatively, but alsoquantitatively, with theoretical predictions (as above).

Rat aortic smooth muscle cell (A10) culture experiments were used todemonstrate whether the magnetic localization techniques have thepotential for therapeutic capabilities. To test for this potential, GFPencoding adenovirus was attached to the nanoparticles and exposed to aconfluent layer of A10 cells grown in culture for 1 minute on top of a316 Stainless Steel Electron Microscopy grid. A number of controlprotocols employing different formulations or magnetic exposureconditions were used to elucidate the importance of various experimentalparameters. The control formulations included Ad added in the presenceof nonmagnetic D1-modified NP, nIgG-modified MNP possessing no specificbinding affinity for the virus, or free Ad. Control experimentalconditions included cell incubation with MNP-Ad complexes in the absenceof either magnetic field or a SS grid. Average and standard deviationswere taken for each group and are graphically illustrated as therelative gene expression with respect to the average free virus control.As demonstrated in FIG. 18C, significant gene expression occurred onlyfor the experimental set, which consisted of a 316L mesh coated by cellsexposed to magnetic nanoparticles in a 500 Oerstad field for 60 seconds,with the virus attached to the particle through the D1 protein. Bycontrast, the signal was substantially lower for the controls whichlacked either the mesh, the field, used non-magnetic particles or freevirus, or were surface coated with nIgG instead of D1. The micrographsshown in FIG. 18A-B demonstrate that GFP expression (green) is localizedaround the mesh (FIG. 18B), and particles (red) are co-localized in theGFP expressing cells (FIG. 18A).

Experimental Results—In Vivo

To validate the concept of magnetic field-facilitated gene vectordelivery in vivo a well-characterized rat carotid model was employed.The same amount of Ad-GFP was locally delivered to the isolatedballoon-injured segments of carotid arteries, either as free vector, orconjugated to biodegradable magnetic NP. The later mode of delivery wascarried out with or without preceding implantation of 316L stainlesssteel spring (modeling prototype stent) in the artery. Transgeneexpression was analyzed in the excised arterial sections 7 days aftergene vector delivery. Fluorescence microscopy of the arterial sectionslocally treated with either free (n=5) or magnetic NP coupled Ad-GFP(the later with (n=6) or without (n=5) magnetisable coilpre-implantation) showed qualitatively much higher GFP expression levelswhen magnetic NP were delivered in conjunction with coil implantation(FIG. 19C vs 19A and 19B).

The invention will be illustrated in more detail with reference to thefollowing Examples, but it should be understood that the presentinvention is not deemed to be limited thereto.

EXAMPLES Example 1 Preparation of Water-Soluble Photo-ActivatablePolymers Based on Poly(allylamine) Synthesis of PAA-BzPh

Synthesis of PAA-BzPh is demonstrated in FIG. 2. Poly(allylamine) (PAA)base was prepared from PAA hydrochloride (Sigma-Aldrich St. Louis, Mo.,MW=70 KDa) by treatment in aqueous medium with a strong anionite DowexG-55 followed by replacement of water by 2-propanol. A 5.1% solution ofPAA base in 2-propanol (4.06 g, containing 3.65 mmol of amino groups)was diluted with CH₂Cl₂ (7 ml) and cooled on an ice bath. N-succinimidyl4-benzoylbenzoate (236 mg, 0.73 mmol; synthesized by applicants andavailable from Toronto Research Chemicals) in CH₂Cl₂ (12 ml) was addedover a 10-min period. The mixture was stirred near 0° C. for 10 min,then warmed to room temperature and acidified with concentrated HCl(0.24 ml, 2.9 mmol). The resulting suspension was dried in vacuo,resuspended in CH₂Cl₂, and the precipitate was filtered off. Afterwashing with CH₂Cl₂ and pentane, 0.544 g of PAA-BzPh hydrochloride wereobtained. A ¹H NMR study of this polymer (utilizing D₂O) indicated that20% of polymer's amino groups were modified with 4-benzoylbenzoicresidues (broad signal at 6.9-8.0 ppm). Analogously, using thecalculated amount of fluorescein isothiocyanate (FITC) (Sigma-Aldrich,St. Louis, Mo.) simultaneously with N-succinimidyl 4-benzoylbenzoate inthe reaction with PAA base, FITC-labeled PAA-BzPh having about 20% of4-benzoylbenzoic residues and about 2% of the FITC label was prepared.

Synthesis of PDT-BzPh

Synthesis of PDT-BzPh is shown in FIG. 2. 5.1% solution of PAA base in2-propanol (2.671 g, containing 2.40 mmol of amino groups) was dilutedwith CH₂Cl₂ (5 ml) and cooled on ice. N-succinimidyl 4-benzoylbenzoate(145 mg, 0.45 mmol) and SPDP (Pierce Biotechnology Inc, Rockford, Ill.,281 mg, 0.90 mmol) were simultaneously dissolved in CH₂Cl₂ (8 ml) andintroduced over a 5-min period. The mixture was stirred near 0° C. for15 min, and succinic anhydride (130 mg, 1.30 mmol) was added at once.The stirring at 0° C. was continued for 0.5 h, the mixture was dried invacuo and extracted first with ethyl acetate and then with water. Thepolymeric residue was dissolved in water (15 ml) with addition of KHCO₃(0.3 g, 3.0 mmol). The solution was filtered and acidified with H₃PO₄ topH of 3.5. The precipitate was filtered off, washed with water, andair-dried PDT-BzPh (488 mg) was obtained. A ¹H NMR study of this polymer(utilizing D₂O and K₂CO₃ at pH 9) indicated that about 40% of2-pyridyldithio groups and about 20% of 4-benzoylbenzoic residues wereattached to the PAA backbone. The rest of amino groups was modified with3-carboxypropionyl residues resulting from succinic anhydride.

Example 2 Preparation of Water-Soluble Photo-Activatable Polymers Basedon Polyacrylic Acid

Polyacrylic acid can be coupled in aqueous solutions simultaneously withamino-derivatized benzophenone compound and a compound (alsoamino-derivatized) containing one of the reactive groups describedabove, particularly 2-(2-pyridyldithio)ethylamine. Water-solublecarbodiimide (EDC) can be used for such coupling (see Scheme below).

Example 3 Photo-Immobilization of Polymeric Modifiers onto Matrix

Surface Amination of Polymeric Matrix with PAA-BzPh.

An aqueous solution (2 mg/ml) of PAA-BzPh or its FITC-labeled variantwas mixed with an equal volume of a buffer containing 0.1M NH₄OAc and0.05M NH₃. PU Tecothane TT-1074A films or polyester (PE) fibers wereimmersed into the mixture for 5-60 min, rinsed with a 1% solution of NH₃and dried on a filter paper. The polymers were irradiated under anUV-lamp (UVGL-25, long wave) for 15-30 min to achieve the covalentbinding of modifiers to the polymer surface. Finally, the surfacemodified polymers were thoroughly washed with diluted (2%) HCl andwater.

Modification of Polymeric Matrix with PDT-BzPh.

PDT-BzPh (30 mg) was dissolved in water (30 ml) by addition of KHCO₃ (20mg) and acidified with a 20% solution of KH₂PO₄ (1 ml). PU films and PEfibers were soaked in the resulting mixture for 5-40 min., rinsed with0.1% acetic acid, dried and irradiated as above. Finally, the polymerswere exhaustively washed with 0.1M KHCO₃ and water.

Example 4

Fluorescent Labeled PAA-BzPh Studies Demonstrating Attachment ofBiomolecules

The presence of modifier bound to the polymer surfaces was confirmed byfluorescence microscopy of PU films and PE fibers surface modified withFITC-labeled PAA-BzPh (pictures are not shown herein).

Example 5

Cell Culture Data Demonstrating Antibody Linkage of Cy3 LabeledGFP-Adenovirus PU Matrix

Surface-aminated PU films (group NH₂-A) were reacted with LC-sulfo-SPDPdissolved in PBS (9 mg/ml; 1 ml; 90 min). Then, the films wereextensively washed in PBS and reacted in 5% BSA with anti-knob Ab (0.66mg/ml) reduced with 1.5 mg of 2-mercaptoethylamine for 90 min at 37° C.Prior to conjugation, Ab was purified by gel filtration using adesalting column equilibrated with degassed PBS containing 10 mM EDTA.The conjugation was allowed to run for 38 hours at room temperature (RT)under mild shaking. Next, the films were washed in PBSx3 and immersed inthe suspension of 10¹¹ particles of Cy3-labeled adenovirus (Cy3-AdV-GFP)in 1.5 ml of 5% BSA/PBS. Surface aminated films that were not modifiedby antiknob Ab (NH₂—B) served as controls. Immunoconjugation was carriedout for 12 hours at RT under mild shaking. Finally, the films werewashed in PBS and examined under fluorescent microscope to assesstethering of Cy3-labeled adenoviruses. A uniform virus coverage of thesurface was observed for the films conjugated with antiknob Ab, whilethe control films were virtually non-fluorescent (images are not shownherein).

PDT-BzPh-modified PU Tecothane TT-1074A films were directly modifiedwith the reduced antiknob Ab. After washing the films (PDT-A) along withthe control samples, the PDT-BzPh-modified PU samples that were notconjugated with the antiknob Ab (PDT-B), both PDT-A and PDT-B wereincubated with Cy3-labeled GFP-AdV. Antibody reduction, purification andconjugation, and virus tethering were carried out according theprocedures outlined above. Similar to the results obtained for thesurface-aminated PU samples, a uniform fluorescent AdV layer wasobserved for the Ab-mediated AdV tethering, while no Cy-3-labeled AdVwas bound to the surface of control films (images are not shown herein).

PE Matrix

PDT-BzPh-modified PE fibers were reacted with 2 mg of antiknob antibodyreduced with 10 mg of 2-mercaptoethylamine at 37° C. for 1 hour. Priorto utilization, the reduced Ab was purified using a desalting columnequilibrated with degassed PBS/10 mM EDTA. The conjugation was carriedout in 5% BSA/PBS for 20 hours at room temperature under mild shaking.

After PBS ×3 washing, the Ab-coupled fibers were immersed into thesuspension of 5×10¹¹ particles of Cy3-labeled adenovirus in 1.5 ml of 5%BSA/PBS. Immune conjugation was carried out for 14 hours at RT undermild shaking Immobilization of Cy3-AdV on the surface ofPDT-BzPh-modified PE fibers was confirmed by fluorescent microscopy(images are not shown herein).

Example 6 Preparation of Nanoparticles (Nps) Surface-Modified withPdt-Bzph

In the following example, an aqueous dispersion of NPs was prepared byemulsification-solvent evaporation and subsequently surface-modifiedwith PDT-BzPh.

D,L-(poly)lactide (D,L-PLA, Sigma), branched poly(ethyleneimine) (PEI25K, Aldrich) and Poloxamer 188 (Pluronic F-68, Sigma) were dissolved in5 ml DCM HPLC grade in amounts of 300 mg, 100 mg and 50 mg,respectively.

Poly(lactide) is a nanoparticle-forming polymer (i.e. constitutes thepolymeric matrix of the nanoparticle). Poly(ethyleneimine) makes thenanoparticles cationic, capable of binding anionic substances andcontributes to nanoparticles' stability during the emulsification stepof their preparation; is not a requisite and can be omitted. Poloxamer188 is a non-ionic stabilizer, which is required for nanoparticles'stability, it provides sterical stabilization.

The organic solution was emulsified in 15 ml of distilled water onice-bath at 0° C. using sonication. The solvent was removed by rotaryevaporation at 35° C. The NPs were filtered through Whatman paper filter(2.5 μm cut-off). The NP size was determined by Photon CorrelationSpectroscopy and was found to be 600 nm. Next, the NPs were dialyzed toremove unbound stabilizers (300 KDa cut-off MW) in distilled water at 4°C. for 48 hr with several water replacements.

2 ml of preformed PLA/PEI nanoparticles were mixed with 1 ml of 0.1%aqueous solution of PDT-BzPh. 50 μl of aqueous solution of 15% KH₂PO₄were added to the reaction mixture to adjust the pH to 5.5. At this pH,PDT-BzPh separates from the solution, presumably associating with thelipophilic surface of the NPs. The total amount of PDT-BzPh in theformulation (1 mg) was calculated to have a 4-fold excess over theestimated amount of the PDT-BzPh needed to establish a monolayer on thesurface of PLA/PEI nanoparticles. The reaction mixture was transferredto a round-bottom flask. The flask was rotated (at 100 rpm) causing thinlayer distribution of the reaction volume over the flask's walls. Thehand-held UV lamp (UVGL-25, UVP) was approximated to the rotating flask,and the reaction mixture was irradiated at about 350 nm from thedistance 1-2 cm for 30 min at room temperature. PDT-BzPh -modified NPs(as described in Example 6) were separated from the excess of PDT-BzPhby the dialysis against double distilled water (DDW) through the 300 kDacut-off membrane (24 hours, 3 changes of DDW).

Example 7 Tethering of Biomolecules to NPs Modified with DPT-BzPh

A thiolated (cysteinated) form of D1 domain of the Coxsackie-Adenovirusreceptor (produced in-house according to the procedure described inNyanguile et al, Gene Ther., 2003; 10:1362-1369) was conjugated to thethiol-reactive NP to impart adeno-virus-tethering properties.

2 mg of D1 was column-desalted in 2.7 ml of degassed PBS supplementedwith 10 mM EDTA and mixed with 3.3 ml of PDT-BzPh-modified NP. Thecoupling reaction was carried out for 14 hours at RT under moderateshaking. D1-derivatized NPs were separated from the excess of D1 by thedialysis against PBS through the 300 kDa cut-off membrane for 36 hoursat 4° C. Finally, a 2 ml aliquot of 6 ml dialyzate was mixed with 125 μlof Cy3-modified Ad-GFP (3.75×10¹¹ particles). BSA was added to final 5%concentration. Immune conjugation was carried out at room temperatureunder mild shaking for 12 hours. 2 ml of diluted (1:3) non-modifiedPLA/PEI NPs were mixed with the same amount of BSA and Cy3-AdV-GFP toserve as control. Tethering of fluorescent Cy3-labeled adenovirus (AdV)to NPs was documented by fluorescent microscopy (images are not shownherein).

Example 8 Preparation of Nanoparticles (Nps) Surface-Modified withPDT-BzPh

An alternative method does not require use of additional stabilizers,such as PEI or Poloxamer 188, relying on the charge stabilization of theNPs provided by the anionic chains of the PDT-BzPh. 200 mg ofpolylactide labeled with BODIPY 564/570 was dissolved in 5 ml chloroformto form an organic phase. 10 mg of PDT-BzPh was dissolved along with 6.7mg potassium bicarbonate in 15 ml water (pH ˜6.5). The organic phase wasemulsified in the aqueous solution by sonication with simultaneousaddition of 1 ml MES (2-[N-morpholino]ethanesulfate) buffer (0.1 M, pH5.5). The organic solvent was removed by evaporation under reducedpressure at 30° C. The obtained particles were filtered through a 1.0 μmglass fiber prefilter (Millipore, Bredford, Mass. USA) and exposed tolong-wave UV for 5 min to achieve covalent attachment of PDT-BzPh. TheNP were separated from the unbound PDT-BzPh by gel filtration (SepharoseB6 gel, Sigma-Aldrich) using MES buffer (0.01 M, pH 6.5) as an eluent.

Surface-activated NP were reacted with thiolated D1 at room temperatureovernight by combining 1.5 ml of NP suspension with 1.5 ml solutioncontaining 0.7 mg protein. Protein-modified NP were separated from theunbound protein by gel filtration and lyophilized with 10% glucose ascryoprotectant. The NP were kept at −20° C. and resuspended in 0.2 mlwater before use.

Replication defective Ad vectors (Type 5; E1, E3 deleted) were obtainedfrom the Gene Vector Core Facility of the University of Pennsylvania(_(GFP)Ad, _(LUC)Ad and _(NULL)Ad) and from the Gene Therapy CoreFacility of the University of Iowa (_(iNOS)Ad). All transgenes wereunder the control of the CMV promoter. Poly(D,L-lactide) (Mw75,000-120,000) was obtained from Sigma-Aldrich (St. Louis, Mo., USA).Mouse anti-knob antibody (IgG) was provided by Selective Genetics (SanDiego, Calif., USA). Non-immune sheep IgG was obtained from Cedarlanelaboratories (Hornby, Ontario, Canada). Recombinant fiber knob proteinwas prepared by cloning the DNA encoding the knob domain into pET15b(Freimuth et al., 1999) and purified as known in the art (see, e.g.,Henry et al., 1994). All chemicals were of analytical grade.

Example 9 Preparation of Nanoparticles (NPs) Surface-Modified with PBPCand PBMC

Poly(allylamine) hydrochloride (Sigma-Aldrich) with number and weightaverage molecular weights of 8500-11000 and ca. 15000, respectively,which on average corresponds to nearly 100 units of monomer for eachmacromolecule, was transformed into poly(allylamine) free base bytreatment with a strongly basic anionite Dowex G-55 in OH-form.Poly(allylamine) was modified at 0° C. in anon-aqueous solvent system(2-propanol/CH₂Cl₂) by acylation of its amino groups with thecorresponding N-hydroxysuccinimide esters to form pendant photoreactive(benzophenone) and either pyridyldithio or maleimide groups. Theunreacted amino groups were quenched with succinic anhydride resultingin pendant carboxylic groups (FIG. 14). The conditions used for themodification virtually eliminated the possibility of side reactions,such as hydrolysis of the active esters. The molar ratio between theattached functional groups could be readily controlled by the amounts ofthe corresponding reagents and was chosen to be 1:2:2 for benzophenone,thiol-reactive and succinamoic residues, respectively). The productsconsisting of poly(allylamine) randomly N-acylated with residues of4-benzoylbenzoic, succinic, and 3-(2-pyridyldithio) propionic or6-maleimidocaproic acids were isolated as free acids and analyzed by ¹HNMR (in D₂O/KDCO₃ or DMSO-d₆ used for the two derivatives abbreviatedPBPC and PBMC, respectively). Aromatic protons of pendant benzophenoneresidues appeared in D₂O/KDCO₃ as a broad band at 6.5-8.0 ppm, whereasin DMSO-d₆ 3 close bands (with maxima at 7.46, 7.68 and 7.98 ppm) wereobserved. Pyridyldithio groups (in D₂O/KDCO₃) showed 3 bands of protonswith maxima at 8.2 (1H), 7.5 (2H) and 7.0 (1H) ppm. Maleimido groups (inDMSO-d₆) exhibited a narrow band with a maximum at 6.93 ppm. CH₂ ofsuccinamoic residues showed up at 2.4 ppm (both in D₂O/KDCO₃ andDMSO-d₆) overlapping with other signals of the polymer's protons.

Surface-activated NP were prepared by a modification of theemulsification-solvent evaporation method (Quintanar-Guerrero et al.,1998; Rosca et al., 2004). In a typical preparation, two hundred mg ofpolylactide covalently labeled with BODIPY 564/570 (Molecular Probes,Eugene, Oreg. USA) was dissolved in 5 ml chloroform to form an organicphase. Ten mg of the surface activating agent synthesized as above wasdissolved along with 6.7 mg potassium bicarbonate in 15 ml water. Theorganic phase was emulsified in the aqueous solution by sonication withsimultaneous addition of 1 ml MES (2-[N-morpholino]ethanesulfate) buffer(0.1 M, pH 5.5). The organic solvent was removed by evaporation underreduced pressure at 30° C. NP were filtered through a 1.0 μm glass fiberprefilter (Millipore, Bredford, Mass. USA) and exposed to long-wave UVfor 5 min to achieve covalent attachment of PBPC or PBMC. The NP wereseparated from the unbound agent by gel filtration (Sepharose B6 gel,Sigma-Aldrich) using MES buffer (0.01 M, pH 6.5) as an eluent.

The human recombinant D1 domain of CAR was prepared as describedpreviously (Nyanguile et al., 2003) and PCT Application Serial No.PCT/US04/026509 by inventors, incorporated herein in its entirety. D1obtained in the form of thio ester was reacted with cysteine (20 mg/ml)and purified by gel filtration using a Polyacrylamide 6000 column(Pierce, Rockford, Ill. USA). Two mg of antiknob antibody or non-immuneIgG was reduced with 5.0 mg of 2-mercaptoethylamine in 1 ml MES buffer(0.01 M, pH 6.5) for 1 hr at 37° C. and purified by gel filtration usingSepharose B6 gel.

Surface-activated NP were reacted with thiolated D1 or reduced IgG atroom temperature overnight by combining 1.5 ml of NP suspension with 1.5ml of a solution containing 0.7 mg protein. The total number ofsurface-associated pyridyldithio groups and the protein-particle bindingwere determined by measuring the characteristic absorbance ofpyridine-2-thione formed in the course of the reaction withdithiothreitol and the protein, respectively (Hermanson, 1996), in theparticle suspending medium (λ=343 nm). Protein-modified NP wereseparated from the unbound protein by gel filtration and lyophilizedwith 10% glucose as a cryoprotectant. The NP were kept at −20° C. andresuspended in deionized water before use. NP size was determined usingphoton correlation spectroscopy (Brookhaven Instruments, Holtville, N.Y.USA).

NP formulated and surface-activated as described above formed acolloidally stable suspension over 14 days with a narrow sizedistribution in the submicron range (305±40 nm). It was observed thatpyridyldithio (PDT) functional groups in the composition of the polymerused for NP surface activation remained stable upon storage under argonat 4° C. maintaining their thiol reactivity for over 14 days. 15% of thePBPC polymer initially used to prepare the formulation was found to beassociated with the NP, corresponding to about 4.5×10⁵ of PDT groups perparticle as calculated from the amount of pyridine-2-thione formed inthe course of the reaction. The reaction with thiolated D1 protein wasrapid, resulting in ca. 3.3×10⁴ of surface-immobilized Ad bindingproteins per particle after 2 hr. A similarly rapid reaction wasobserved with reduced non-immune IgG used for surface modification ofcontrol particles (results not shown). The modification of the NP withthe proteins of interest led to an increase in the particle size from305±40 nm to 409±55 nm and 457±53 nm for the D1-coated NP (_(D1)NP) andnon-immune IgG-coated NP (_(nIgG)NP), respectively. Transmissionelectron microscopy confirmed formation of _(D1)NP-Ad complexes witheach individual nanoparticle capable of immobilizing numerous viralunits on its surface (see FIGS. 8A-8B).

Example 10 AdV Immobilization on the NP Surface See FIGS. 5, 6A-C and 7A-F Surface Activation Agent Requirements:

-   -   1. preferably localizes on the particle surface (amphiphilic);    -   2. capable of multiple point covalent surface-attachment to        provide stable NP-AdV association; and    -   3. bears thiol-reactive groups minimally affected by a        short-term UV exposure.        Fluorescently labeled 370 nm NP and AdV incubated for 2 hr at        1:1 ratio in MES buffer, pH 6.5. Magnification is ×200. Note the        substantial co-localization of the signal. Transmission electron        micrograph shows a composite negatively stained with 2% uranyl        acetate.        Fluorimetry and fluorescent microscopy of live cells:    -   Smooth muscle cells (A10), blood origin endothelial cells (BOEC)        and heart endothelioma cells (H5V) were seeded at 104/well on        96-well plates. GFP or inducible NO synthase (iNOS) encoding AdV        was incubated for 1 hr with varying concentrations of non-immune        IgG- or D1-coated NP (0-4.0 μg PLA or 0-1.5×10⁸ particles/well)        and applied to the cells in 10% FBS-supplemented DMEM for 2 hr.        NP uptake:    -   NP were labeled with BODIPY 564/570; measured using RED        fluorescence channel (544/580).        Transduction efficacy:    -   GFP was determined using GREEN fluorescence channel (485/535).        Cell appearance:    -   Fluorescent microscopic observation

The effect of knob fiber protein on the transduction and uptake ofcomposite-forming NP measured as a function of NP dose in smooth musclecells (A10).

The cells were pretreated with knob (5 μg/ml) for 1 hr prior to additionof AdV (2×108/well) in the presence of D1NP (0-4 μg PLA/well), or nIgGNPused as a control. Knob containing cell medium was aspirated; the cellswere washed with PBS and incubated for 2 hr with the formulations. Geneexpression was assayed 2 days post treatment.

Biodegradable polymer based NP-AdV composites were formulated using avector-specific affinity attachment strategy (with D1). The compositeswere effectively taken up by vascular cells via a CAR-independentpathway resulting in a potent gene transfer in vitro. No non-specificcell toxic effects were associated with the composites in the studieddose range. The ability to deliver therapeutic genes was demonstratedusing composites formulated with iNOS AdV that significantly inhibitedgrowth of smooth muscle cells in good correlation with the GFP reporterexpression. The growth inhibitory effect is of relevance for genetherapy of cardiovascular disease and needs further evaluation in vivo.

Example 11 Cell Culture Experiments with NP

Rat aortic smooth muscle cells (A10) and murine heart endothelioma cells(H5V) were grown in DMEM medium supplemented with 10% fetal calf serum(FCS). Sheep blood outgrowth endothelial cells were obtained fromfreshly drawn peripheral blood (Lin et al., 2000) and cultured in EGM-2medium (Cambrex, East Rutherford, N.J.) supplemented with 5% fetal calfserum (Cambrex) on tissue culture dishes precoated with acetic aciddenatured collagen. The cells were seeded on clear bottom 96-well platesat a density of 104 or 3×103 cells/well for reporter expression andgrowth inhibition studies, respectively. NP were combined with Ad in MESbuffer (0.01 M, pH 6.5) containing 5% albumin to prevent non-specificbinding, left on a shaker for 1 hr at room temperature, then dilutedwith respective medium at a volume ratio of 1:4. The cells wereincubated with the formulation of interest (100 μl/well) for 2 hr andthe medium was replaced after washing with PBS. NP uptake and GFPexpression were assayed fluorimetrically in live cells using λem/λex of485 nm/535 nm and 544 nm/580 nm, respectively. In the knob competitionexperiment A10 cells were incubated for 2 hr with DINP-Ad or equivalentamounts of Ad and nIgGNP used as a control (2×108/well and 0-4 μgPLA/well, respectively) after pretreatment with 5 μg/ml knob for 1 hr.Growth inhibition of A10 cells was determined using the Alamar Blueassay as described by the manufacturer (Biosource, Camarillo, Calif.USA). All cell culture experiments were carried out in triplicates.

Example 12 The Extent of Gene Transfer by NP-Ad Complexes In Vitro

To determine the effect of Ad association with NP on the uptake of thecomplexes and level of gene transfer in different cell types, the NP-Adwere added to cultured rat smooth muscle cells (A10), sheep endothelialprecursor cells (BOEC) and murine endothelioma cells (H5V). The lattercell type was chosen as an example of a CAR-deficient cell line (Ogawaraet al., 2004). The cellular uptake of D1NP-Ad was proportional to the NPdose, and was equally efficient in A10 and BOEC cells, being slightlyhigher for H5V (FIGS. 9 A and B). The amounts of the NP resident insidethe cells decreased gradually over 72 hours with comparable NP retentionlevels averaging for all cells types 61±3% and 54±4% of the initiallymeasured amounts for the D1NP and nIgGNP, respectively, administered ata dose of 1.6 μg PLA/well (FIG. 9 B). Ad immobilization on D1NPeffectively increased gene transfer in all types of cells in a NP dosedependent fashion in comparison to free Ad (p<0.001) or Ad in thepresence of control nIgGNP (p<0.001, FIGS. 7 A-F) despite the comparablyhigh internalization of the two NP types, suggesting a lack of stableNP-Ad association in the control formulation. The transduction rates bythe NP-Ad correlated with the permissivity for gene transfer by free Adin the three cell types, with BOEC and H5V exhibiting the highest andthe lowest transduction efficiences, respectively. While the amount ofthe GFP continued to increase over a 72 hr period in A10 and BOEC (FIGS.7 D, 7E), the gain in the extent of gene transfer mediated by D1NP-Ad(expressed as the fold increase in comparison to cells treated withequal amounts of nIgGNP and Ad) was the highest 1 day post treatment inthese cell lines (5.3- and 3.4-fold, respectively, at 1.6 μg PLA/well).In contrast, GFP expression in the H5V cells treated with D1NP-GFPAdcontinuously increased in a near-linear fashion for 7 days (results notshown), while the reporter levels in the control cells treated withnIgGNP remained slightly above the detection limit of the assay (FIG. 7F).

The effect of knob protein on cellular uptake and gene transfer by NP-Adcomplexes will now be described.

Pretreatment of cultured smooth muscle cells with knob protein resultedin a substantial inhibition of gene transfer by Ad applied in thepresence of control nIgGNP, as well as free Ad (53±4%, FIGS. 6 A, 6B) inagreement with its receptor-dependent cell entry. In contrast, when Adwas administered with D1NP (FIGS. 6A-C), the inhibitory effect of theknob pretreatment on the transduction decreased with the NP dose (13±8%at 4.0 μg PLA/well), corresponding apparently to an increase in thefraction of Ad transported into the cells in a NP surface-immobilizedform. These results support the view that cellular uptake of the NP-Adinvolves a CAR-independent transport mechanism. Receptor-uncoupledtransport of the Ad formulated in D1NP-based complexes is also supportedby similar rates of the D1NP uptake by smooth muscle cells with orwithout knob pretreatment (FIG. 6 C).

The effect of the type of NP-Ad binding will now be described.

The NP-Ad complexes taken up by cells would come in contact with thereducing environment of the cell interior (Saito et al., 2003) where thedisulfide linkage of the Ad binding protein molecule to the particlesurface could hypothetically be cleaved to release the virus from thecarrier particle and allow it enter the intracellular processing leadingeventually to the expression of the gene. Alternatively, the entry ofthe virus into the intracellular processing pathway may involve thedissociation of the affinity bond between the Ad binding protein and theknob protein of the virus, or it may occur through decomposition of thevirion shell. To elucidate the significance of the cleavable disulfidebond that links either D1 or antibody to NP, we used the PBMC derivativeincorporating maleimido (MI) groups that form non-biodegradable C—Sbonds upon reaction with thiols, but otherwise is analogous to the PBPCsurface modification that results in PDT-based derivatization (FIG. 10A). The NP modified with either of the two polymers was formed withcomparable size distributions (305±40 nm and 303±36 nm for PDTNP andMINP, respectively). Internalization of the NP-Ad prepared withMI-activated NP was comparable to that observed with the PDT derivativeboth in terms of efficiency and the linear pattern of the dependence onthe particle dose (FIG. 10 B). In accordance with the similarlyefficient cellular uptake, a comparable time course of gene expressionand transduction rates were exhibited by the two formulations (FIGS. 10C and 10 D) with MINP being slightly superior in efficiency at higher NPdoses, suggesting that the biodegradable linkage between the NP and theAd binding protein does not contribute significantly to the mechanismresponsible for the potentiation of gene transfer by NP-Ad.

Virus immobilization using D1 vs. anti-knob antibody will now bedescribed.

A knob-specific monoclonal IgG antibody that has previously been shownby our group to provide affinity-based attachment of Ad to solidsurfaces with effective delivery of the vector in vitro and in vivo(Klugherz et al., 2002), was investigated here in comparison to D1 forthe surface immobilization of Ad on biodegradable NP. While controlnIgGNP were unable to potentiate viral gene transfer at any dose as wasshown in another series of experiments (FIGS. 7A-F), anti-knobIgG-coated NP (AKNP) applied at 0.8 mg PLA/well increased the geneexpression in A10 cells by a factor of 7 to 11 (FIG. 11A) compared tofree Ad. The greatest increase in gene transfer efficiency was achievedat the lowest dose of Ad applied (8.4×107/well). The transduction levelsof the AKNP-based formulation amounted to 50% to 80% of that of D1NP atthe above particle concentration (FIG. 11B). However, in contrast to D1,the potentiating effect of the AKNP decreased for the higher NP doses(1.6-4.0 μg PLA/well) resulting in a poor dose dependence (p<0.001 andp=0.97 for D1NP and AKNP, respectively). The decrease in efficiency atparticle doses higher than 1.6 μg PLA/well was associated with theextensive formation of large-sized aggregates (data not shown) that wasdetected by fluorescent microscopic examination and was not observed inthe presence of either D1NP or nIgGNP. The NP aggregation was triggeredby addition of Ad manifesting colloidal destabilization of the carrierparticles upon binding increasing amounts of Ad, and this stronglyaffected the cell entry of the complexes apparently resulting insuboptimal intracellular levels of the gene vector. Additionally, therate of Ad entering its intracellular processing followinginternalization might be compromised by entrapment in aggregatedparticle clusters.

A substantially larger amount of perinuclearly localized D1NP wasobserved in all cells 24 hours post treatment in comparison to AKNP(FIG. 12 (samples A and B)), the latter also showing a less uniformdistribution between the cells. The level of the gene expression wascorrespondingly higher for D1NP-Ad than for AKNP-Ad formed at thehighest NP dose (FIG. 12 (samples D and E)), while cells treated withfree Ad showed the lowest transduction rates (FIG. 12 (sample F)).

The effect of NP-iNOSAd complexes on arterial smooth muscle cell growthwill now be described.

The extent of iNOSAd-mediated A10 cell growth inhibition wassignificantly greater with D1NP as compared to control nIgGNP (p<0.001and p=0.07, respectively) amounting to 66% and 22% at the highest testedamounts of the NP and Ad (FIGS. 4 A, 4 B) per cell proliferation assayresults. In addition, cell growth was examined following treatment withthe GFPAd- and null Ad-carrying formulations (FIG. 4 C, 4 D). Thesecontrols were included in order to estimate the effect of the complexesmediating transfer of a gene with no specific cell inhibitory activity,as well as the effect of the formulation per se in the absence of geneexpression. While the cell growth was not strongly affected by the NPdose of the null vector-based formulation (p=0.19), a substantial cellgrowth inhibition increasing with the NP dose was evident followingtreatment with NP-GFPAd (p<0.001), and could be attributed to the geneproduct expressed and accumulated at high levels in the cells. Thenonspecific effect of GFP on the rate of cell growth however remainednotably lower than that of iNOSAd associated with higher doses of D1NP(23% vs. 66%, respectively, at the highest tested amounts of the NP andAd). The strong direct dose dependence of the cell growth inhibition onboth NP and Ad exhibited by the D1NP-iNOSAd, and the notably weakerdependence on the particle dose observed in the presence of nIgGNP wereparalleled by the GFP expression pattern (FIGS. 4 E, 4 F): a direct dosedependence was characteristic of D1NP with saturation reached at aparticle dose of 3.2 μg PLA/well (p<0.001), while control NP had nosignificant potentiating effect (p=0.08).

Example 13 In Vivo Gene Expression

Animal procedures were performed in compliance with NIH standardspertaining to the care and use of laboratory animals utilizing aprotocol approved by the I.A.C.U.C. of The Children's Hospital ofPhiladelphia. Sprague-Dawley rats (male, 100-110 g) were anesthetizedusing a mixture of ketamine and xylazine (80 mg/kg and 5 mg/kg,respectively) followed by a subcutaneous injection in dorsal caudalregion with a 100 μl suspension containing either free LUCAd (n=5, 8×109viral particles per animal) or D1NP-LUCAd complex (n=4, 8×109 viralparticles combined with DINP at a dose of 150 μg PLA per animal). Oneand five days post treatment the animals were anesthetized, injected inthe tail vein with luciferin (60 mg/kg) and the bioluminescence was bothimaged and quantitatively measured using the IVIS 100 imaging system(Xenogen Corporation, Alameda, Calif. USA) 15 minutes after injectionwith a signal acquisition time of 20 sec.

_(D1)NP-_(LUC)Ad complexes subcutaneously administered to rats resultedin a localized luciferase expression that was 3.7-fold higher than thatproduced by free _(LUC)Ad 1 day post treatment (p=0.016, FIG. 13 A). Theexpression at one day was the strongest at the injection sites and had asimilar concentric spatial distribution in the _(D1)NP-_(LUC)Ad and free_(LUC)Ad treated animals. At 5 days _(LUC)Ad expression was atcomparable levels in both free Ad and Ad-NP groups; it is of interestthat this reflected a 16-fold reduction in the Ad-NP expression levelnoted at 1 day. This most likely reflects mobilization of the transducedcells in the subcutaneous injection site during the period between the 1and 5 day data acquisition points.

Example 14 Preparation of Magnetic Particles

Magnetically-responsive particles can be prepared similarly to theprocedure described in Examples 6 and 7 using ultra small iron oxidenanocrystals dispersion in chloroform instead of pure chloroform asstated in the procedure above. Such dispersion can be obtained byprecipitation of aqueous ferrous chloride or its co-precipitation withferric chloride in an alkaline aqueous solution and further stabilizedwith a fatty acid (See De Cuyper M, Joniau M. Magnetoliposomes.Formation and structural characterization. Eur Biophys J 1988; 15:311-9;Khalafalla S E. Magnetic fluids, Chemtech 1975, September: 540-7) thatalso imparts a degree of lipophilicity to the nanocrystal surfacedepending on the hydrocarbon chain length and the coating density. Suchcoated nanocrystals can further be extracted into or re-suspended inchloroform as well as other organic solvents, such as dichloromethane,tetrahydrofuran, acetone etc., which can be used for polymer-basedparticle formulation by the methods mentioned above.

Example 15 Delivery of Magnetic Particles bearing Biomaterial

Magnetic Particle Materials were as follows: Poly(D,L-lactide) (MW75,000-120,000), (poly)allylamine hydrochloride (MW 15,000), oleic acid,sepharose beads (45-165 μm), iron (II) chloride tetrahydrate and iron(III) chloride hexahydrate were purchased from Sigma-Aldrich (St. Louis,Mo., USA). BODIPY 564/570 succinimidyl ester was purchased fromMolecular Probes (Eugene, Oreg., USA). All other reagents were ofanalytical grade.

Magnetic Implant Materials were as follows: Palmaz-Shatz Stents composedof 316L grade stainless steel were obtained from Cordis (Warren, N.J.).The stents were approximately 1.5 cm in length, and 2.7 mm in diameterin the unexpanded state. The wire struts had cross-sectional dimensionsof approximately 300 μm wide and 100 μm thick. Electron microscopy gridscomposed of 316 grade stainless steel (E-0200) were purchased fromElectron Microscopy Sciences (Hatfield, Pa.). The electron microscopygrids consisted of a mesh of wires having cross-sectional dimensions ofapproximately 40 μm wide and 20 μm thick and having a 120 μm pitch.

Electroplating materials were as follows: cobalt (II) chloridehexahydrate, nickel (II) chloride hexahydrate, saccharin, and boric acidwere purchased from Sigma Aldrich. A model 363 Potentiostat/Galvanostatwas purchased from AMETEK Princeton Applied Research (Oakridge, Tenn.)for current control of up to 1 Ampere.

Cell Culture Materials were as follows: phosphate buffer saline (PBS)with Ca and Mg, phosphate buffer saline (PBS) without Ca and Mg,trypsin-EDTA 1×, and Fetal Bovine Serum (FBS) were purchased fromInvitrogen, Inc. (Grand Island, N.Y.), DMEM 1× was purchased fromMediatech, Inc. (Hernandon, Va.).

Nanoparticles were prepared as follows. A thiol- and photoreactivepolymer, PBPC, for the nanoparticle surface chemical activation wassynthesized. Magnetic nanoparticles were prepared by a modification ofthe emulsification-solvent evaporation method. Ferrous and ferricchlorides were dissolved in water, and mixed iron oxide was obtained byprecipitation with 1 N sodium hydroxide. Oleic acid was added to themixture containing the precipitate, and heated to 90° C. in water bathfor 5 min with stirring. The precipitate was washed with ethanol andresuspended in chloroform. Poly(D,L-lactide) covalently labeled withBODIPY 564/570 was dissolved in the mixed iron oxide suspension inchloroform to form an organic phase. The organic phase was emulsified inthe aqueous solution containing PBPC. Subsequently, the chloroform wasevaporated under reduced pressure; the particles were filtered through a1.0 μm glass fiber prefilter, and irradiated for 5 min with long-wave UVto effect covalent surface attachment of PBPC to make nanoparticlesthiol-reactive. The thiol-reactive were separated from the unboundpolymer by gel filtration on sepharose gel.

Nanoparticle modification with recombinant D1 protein: Human recombinantD1 domain of the Coxsackie-adenovirus receptor was prepared as describedabove and reacted in the form of thioester with cysteine (20 mg/ml) toobtain thiolated D1. 2 mg of sheep non-immune immunoglobulin (nIgG)obtained from Cedarlane laboratories (Hornby, Ontario, Canada) and usedas a control was reduced with 2-mercaptoethylamine (5 mg) in 1 ml IVIESbuffer (0.01 M, pH 6.5); both proteins were purified by gel filtration.Thiol-reactive NP were reacted overnight with thiolated D1 or reducednIgG (0.7 mg) by combining 1.5 ml of NP suspension with 1.5 ml proteinsolution. Protein-coated NP were separated from free protein by gelfiltration and lyophilized with 10% glucose as cryoprotectant. The NPwere kept at −20° C. and resuspended in 0.2 ml water before use. NP sizewas determined using photon correlation spectroscopy (BrookhavenInstruments, Holtville, N.Y. USA).

Stent Metallization: A solution of 0.8 M NiCl₂, 0.25 M CoCl₂, 30 g/LH₃BO₃, and 1 g/L Saccharin was prepared at a pH of 3. An electroplatingbath containing 400 mL of the stock solution was heated to 60° C., and a0.05 Ampere current (corresponding to ˜2 A/mm² current density) waspassed through the stent (having roughly 2 cm² surface area) for a totalof 5 minutes using Cobalt sheet metal as the counter electrode. About10% of the stent's original weight was added by this process.

Stent-Nanoparticle Magnetic Uptake: The Palmaz-Shatz stents were mountedon a BxVelocity balloon expandable stent system obtained from Cordis(Miami Lakes, Fla.) and expanded into 3.2 mm diameter tubes purchasedfrom United States Plastic Corp (Lima, Ohio). A stock vial of lypholizedmagnetic nanoparticles was dissolved in 0.05 M Hepes buffer containing0.9% NaCl, 5% albumin, and adjusted to pH 7.4. One fifth of the stockvolume was injected into the tube nearby the stent, and the tube wasexposed to uniform 1000 Oerstad field for 60 seconds by holding the tubein between two solenoid coils with iron cores. After sixty seconds, thestents were explanted from the tubes, cut open, pressed flat en faceonto a glass slide, and imaged in a Nikon Eclipse TE300 Microscope. The535/580 channel was used to view the BODIPY label incorporated into thenanoparticles. Six representative stent wires were selected for eachgroup. A histogram reading was employed to quantify the averageluminosity of each wire in the photograph and the values were subtractedfrom the background in the same photograph. The stent wires in eachphotograph have planar dimensions of 210 μm by 860 μm. As a reference,the original stock volume of nanoparticles was diluted one hundred fold,and then 24 was dispensed into a glass slide and allowed to evaporate.The average luminosity of a representative area of the drop, equivalentto the region used to image the stent wire, was quantified and comparedto the luminosity of the experimental groups. The total area of theevaporated drop was 5.3 mm², which allowed for quantification of thefraction of injected nanoparticles that were trapped by the stent wire.The total surface area of the stent was estimated to be 2.5 cm² bycounting the number of wires and surface area for each wire in thestent. By this method, the fraction of captured nanoparticles wasdeduced from the original amount injected.

Nanoparticle-Magnetic Mesh: A sterilized mesh was placed in each well ofa 24 well plate, and 500 μL of rat aortic smooth muscle cells (A10cells; ATCC, Gaithersburg, Md.) at Passage 68 were seeded at aconcentration of 10⁵ cells/mL onto the mesh at the bottom of the 24 wellplate. The cells were allowed to attach to the mesh and substrate for 72hours. After reaching confluency, 2.0 μl of replication defective Type 5(E1, E3 deleted) GFP-encoding adenovirus obtained from the Gene VectorCore Facility of the University of Pennsylvania (5.3×10¹²/ml) was mixedwith MES buffer (0.01 M, pH7.4) containing 10% albumin. Then, 200 μl ofthe nanoparticles (surface-modified with D1 or nIgG as a control, weredispersed in 1.6 ml MES buffer. The two were mixed (typically 0.5 ml ofvirus with 0.5 ml of particles) and incubated at room temperature for 30min. Finally, 4.0 ml medium supplemented with 12.5% FBS was added toeach particle preparation. The groups (triplicates) were exposed to theAd-nanoparticle complexes for 1 minute in the conditions noted below andthe medium was replaced with fresh DMEM after washing the cells 3 timesto remove the complexes. Then, the 24 well plates were placed in theincubator and periodically evaluated for GFP gene expression.

Fluorescent Microscopy: The smooth muscle cell cultures were rinsed eachday with PBS with Mg & Ca, and each well was mapped in the SpectraMaxGemini EM Fluorescent Scanner (Molecular Devices, Sunnyvale, Calif.).The wells were divided into 1 mm square sections (9 by 9) and scannedusing 485/510 excitation and emission wavelengths. The maximumfluorescent signal in each well was averaged for each of the groups andmapped as a function of time.

Local Arterial Delivery in Rat Model: Male Sprague-Dawley rats (450-500g) underwent a denudation angioplasty injury of the common carotidartery with a 2F Fogarty catheter. The animals were then randomized intothree groups: 1) Ad-GFP intraluminal delivery with no magnetic fieldapplied (n=5), 2) Ad-GFP/magnetic NP delivery+magnetic field (n=5), and3) Ad-GFP/magnetic NP delivery with pre-implanted 316 L stainless steelspring+magnetic field (n=6). In all groups delivery with or withoutmagnetic field enhancement was carried out for one minute into isolatedFogarty-injured segment of common carotid artery. After delivery in thegroups 2 and 3 a magnetic field (150 Gauss) was applied for the first 10mm of reperfusion.

To prepare gene vectors for local delivery 4.5×10¹⁰ particles of Ad-GFPwere mixed with 200 μl of reconstituted suspension of lyophilizedmagnetic NP-D1. For control purposes (group 1) the same amount of Ad-GFPwas diluted in 200 μl of 0.01 M MES buffer.

The animals were sacrificed on the day 7 after delivery. After coilremoval (where applicable), the arteries were embedded in OCT, cut andexamined by fluorescence microscopy.

Statistical Methods: Data are expressed as means±standard error(mean±se). The significance of differences between means of experimentgroups was determined using Student t tests.

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While the invention has been described in detail and with reference tospecific examples thereof, it will be apparent to one skilled in the artthat various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

1-21. (canceled)
 22. A composition comprising a monomolecular layer of awater-soluble photo-activatable polymer and a matrix having at least onecarbon, wherein the photo-activatable polymer comprises (a) aphoto-activatable group, wherein the photo-activatable group is adaptedto be activated by an irradiation source and to form a covalent bondbetween the water-soluble photo-activatable polymer and a matrix havingat least one carbon; (b) a reactive group, wherein the reactive group isadapted to covalently react with a biomaterial; (c) a hydrophilic group,wherein the hydrophilic group is present in an amount sufficient to makethe water-soluble photo-activatable polymer soluble in water; and (d) apolymer precursor; wherein the water-soluble polymer is represented by aformula:

wherein n is 50 to 2000, k is 10 to 1000, and m is 10 to 1000; andwherein the monomolecular layer is attached to the matrix by a covalentbond between the photo-activatable group and the at least one carbon.23. (canceled)
 24. The composition of claim 22, further comprising abiomaterial having a plurality of active groups, wherein the biomaterialis covalently attached to the monomolecular layer by covalent bondingbetween the active groups and reactive groups.
 25. The composition ofclaim 24, wherein at least one of the active groups is a member selectedfrom the group consisting of amine, carboxyl, hydroxyl, thiol, phenol,imidazole, and indole.
 26. The composition of claim 24, wherein the atleast one of the active groups comprises thiol.
 27. The composition ofclaim 24, wherein the biomaterial comprises a member selected from thegroup consisting of an antibody, a viral vector, a growth factor, abioactive polypeptide, a polynucleotide coding for the bioactivepolypeptide, a cell regulatory small molecule, a peptide, a protein, anoligonucleotide, a gene therapy agent, a gene transfection vector, areceptor, a cell, a drug, a drug delivering agent, nitric oxide, anantimicrobial agent, an antibiotic, an antimitotic, dimethyl sulfoxide,an antisecretory agent, an anti-cancer chemotherapeutic agent, steroidaland non-steroidal anti-inflammatories, hormones, an extracellularmatrix, a free radical scavenger, an iron chelator, an antioxidant, animaging agent, and a radiotherapeutic agent.
 28. The composition ofclaim 27, wherein the biomaterial is at least one of an anti-knobantibody, an adenovirus, a D1 domain of the Coxsackie-adenovirusreceptor, insulin, an angiogenic peptide, an antiangiogenic peptide,avidin, biotin, IgG, protein A, transferrin, and a receptor fortransferrin.
 29. The composition of claim 22, wherein the matrix is amember selected from a group consisting of poly(urethane), poly(ester),poly(lactic acid), poly(glycolic acid), poly(lactide-co-glycolide),poly(ε-caprolactone), poly(ethyleneimine), poly(styrene), poly(amide),rubber, silicone rubber, poly(acrylonitrile), poly(acrylate),poly(methacrylate), poly(alpha-hydroxy acid), poly(dioxanone),poly(orthoester), poly(ether-ester), poly(lactone), mixtures thereof andcopolymers of corresponding monomers.
 30. The composition of claim 29,wherein the matrix further comprises a magnetic field-responsive agent.31. The composition of claim 30, wherein the magnetic field-responsiveagent is a superparamagnetic agent selected from the group consisting ofmagnetite and maghemite nanocrystals.
 32. The composition of claim 22,wherein the matrix is an implantable device.
 33. The composition ofclaim 32, wherein the implantable device comprises at least one memberselected from the group consisting of poly(urethane), poly(ester),poly(lactic acid), poly(lactide-co-glycolide), poly(ε-caprolactone),poly(ethyleneimine), poly(styrene), poly(amide), rubber, siliconerubber, poly(acrylonitrile), poly(acrylate), poly(methacrylate),poly(tetrafluoroethylene), organosilane, mixtures thereof and copolymersof corresponding monomers.
 34. The composition of claim 22, wherein thematrix is a particle having a diameter of about 5 nm to about 10microns.
 35. The composition of claim 34, wherein the particle comprisesat least one member selected from the group consisting of poly(lacticacid), poly(glycolic acid), poly(lactide-co-glycolide), polyε-caprolactone), poly(ethyleneimine), poly(lactone), mixtures thereofand copolymers of corresponding monomers.
 36. The composition of claim35, wherein the particle further comprises a biomaterial having aplurality of active groups, wherein the biomaterial is covalentlyattached to the monomolecular layer by covalent bonding between theactive groups and reactive groups.
 37. The composition of claim 36,wherein the biomaterial comprises a member selected from the groupconsisting of an antibody, a viral vector, a growth factor, a bioactivepolypeptide, a polynucleotide coding for the bioactive polypeptide, acell regulatory small molecule, a peptide, a protein, anoligonucleotide, a gene therapy agent, a gene transfection vector, areceptor, a cell, a drug, a drug delivering agent, nitric oxide, anantimicrobial agent, an antibiotic, an antimitotic, dimethyl sulfoxide,an antisecretory agent, an anti-cancer chemotherapeutic agent, steroidaland non-steroidal anti-inflammatories, hormones, an extracellularmatrix, a free radical scavenger, an iron chelator, an antioxidant, animaging agent, and a radiotherapeutic agent.
 38. The composition ofclaim 37, wherein the biomaterial is at least one of an anti-knobantibody, an adenovirus, a D1 domain of the Coxsackie-adenovirusreceptor, insulin, an angiogenic peptide, an antiangiogenic peptide,avidin, biotin, IgG, protein A, transferrin, and a receptor fortransferrin.
 39. The composition of claim 38, wherein the biomaterial isiNOS-AdV or GFP-AdV.
 40. A method of making the composition of claim 22the method comprising: providing the matrix having at least one carbon;providing an aqueous solution of the water-soluble photo-activatablepolymer having the photo-activatable group and the reactive group;contacting the matrix with the aqueous solution; and photo-activatingthe photo-activatable group by irradiation to covalently attach thewater-soluble polymer via the photo-activatable group to the matrix andthereby forming the monomolecular layer of the composition.
 41. Themethod of claim 40, wherein the irradiation is performed at a wavelengthfrom about 190 to about 900 nm.
 42. The method of claim 41, wherein theirradiation is performed at a wavelength of 280 to 360 nm.
 43. Themethod of claim 40, further comprising: providing a biomaterial having aplurality of active groups; and reacting the plurality of active groupswith the water-soluble photo-activatable polymer to covalently attachthe biomaterial to the matrix.
 44. The method of claim 40, wherein thematrix is an implantable device.
 45. A method for delivery of abiomaterial to a cell, the method comprising: contacting the compositionof claim 22 with a biomaterial having a plurality of active groups underconditions sufficient to attach the biomaterial to the monomolecularlayer by covalent bonding between the active groups and the reactivegroups; and administering the matrix to the cell and thereby deliveringthe biomaterial.
 46. The method of claim 45, wherein the biomaterial isat least one of a protein, a D1 domain of the Coxsackie-adenovirusreceptor, an adenovirus, or an antibody specifically bound to a nucleicacid.
 47. The method of claim 45, wherein the matrix is an implantabledevice.
 48. The method of claim 45, wherein the matrix is a particlehaving a diameter of about 5 nm to about 10 microns.
 49. The method ofclaim 48, wherein the biomaterial is iNOS-AdV or GFP-AdV.
 50. A methodfor delivery of a biomaterial to a cell or a tissue, the methodcomprising: providing a bioactive magnetic particle comprising (1) thecomposition of claim 24, wherein the matrix is in a shape of a particlehaving a diameter of about 5 nm to about 10 microns, and (2) a magneticfield-responsive agent associated with the matrix; providing an implantcomprising a magnetic surface to the cell or the tissue; administeringthe bioactive magnetic particle to the cell or the tissue; and capturingthe bioactive magnetic particle onto the magnetic surface, and therebydelivering the biomaterial.
 51. The method of claim 50, wherein deliveryof the biomaterial to a cell or a tissue comprises manipulating at leastone of a particle diameter, proximity of an external magnetic field, adegree of surface magnetization of the implant, and a time interval forcapturing the particle onto the magnetic surface.